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11-15-2010
Design, Synthesis & Biological Activity of Novel Protein Tyrosine Design, Synthesis & Biological Activity of Novel Protein Tyrosine
Phosphatase (PTP) Mimetics Phosphatase (PTP) Mimetics
Sridhar Reddy Kaulagari University of South Florida
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Design, Synthesis & Biological Activity of Novel Protein Tyrosine
Phosphatase (PTP) Mimetics
by
Sridhar Reddy Kaulagari
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy Department of Chemistry
College of Arts and Sciences University of South Florida
Major Professor: Mark McLaughlin, Ph.D. Wayne Guida, Ph.D.
Roman Manetsch, Ph.D. Xiao (Sheryl) Li, Ph.D.
Date of Approval: November 15, 2010
Keywords: Protein Tyrosine Phosphatase, Inhibitors, Pyrimidines, alpha beta epoxy carboxylates, Cysteine Peptide Nucleic Acids (CPNAs)
© Copyright 2010, Sridhar Reddy Kaulagari
DEDICATION
To my grand father late Shri Ramreddy Kaulagari
To my mom Lalitha and dad Anjireddy
To my wife Sandhya and sisters Sukanya and Sudha
To my mentor Mark and all my teachers
To all my good friends and Well wishers
ACKNOWLEDGEMENTS
I am unremittingly thankful to my advisor Dr. Mark L. McLaughlin,
Ph.D, for steering me in each and every step of actualizing this degree.
I am very thankful to my committee members Wayne Guida, Ph.D,
Roman Manetsch, Ph.D, and Xiao (Sheryl) Li, Ph.D for their guidance
towards the completion of my degree. I would like to thank Prof. Jerry
Wu and his group members for performing biological activity studies. I
would like to thank Dr. Pasha Khan for chairing my Defense
Committee. I will never forget the inspiration I received from Venkata
Ramana and Madhusudhan Rao during my junior college years and
working period at Cadila Pharmaceuticals respectively. I would like to
express my sincere gratitude towards my friends and lab mates Sung
Wook Yi, Priyesh Jain, and Fenger Zhou for helping me conduct some
of the experiments and survive through the ones that failed. I thank
Mehul, David, Mingzhou, Laura, Melissa, Phil and Li who shared time
with me during my stay at University of South Florida. I would also like
to acknowledge all the undergraduate students who worked in the lab
for making the laboratory work a fun experience. I would like to
extend my thanks to Ted Gauthier, Ph.D and Edwin Rivera, Ph.D for
helping me in obtaining some the finest data from the analytical
facilities at the university.
I would like to thank all the people in chemistry office who helped me
from day one since I joined the university.
At the end I would like to thank my family and friends for everything
else.
TABLE OF CONTENTS
LIST OF FIGURES ....................................................................... iv
LIST OF SCHEMES ..................................................................... vi
LIST OF TABLES ....................................................................... viii
LIST OF SYMBOLS AND ABBREVIATIONS ....................................... ix
ABSTRACT ............................................................................... xiii
CHAPTER ONE: PROTEIN PHOSPHATASE INHIBITORS ...................... 1
1.1 General introduction to phosphorylation ......................... 1
1.2 Protein phosphorylation ............................................... 3
1.3 Occurrence of protein phosphorylation ........................... 5
1.4 Protein phosphatases and their importance .................... 6
1.5 Protein tyrosine phosphatases (PTPs) ............................ 7
1.6 Phosphotyrosine mimetics ........................................... 9
1.7 Phosphatase inhibitors .............................................. 10
1.8 Challenges in designing a potent and specific
phosphatase inhibitor ................................................ 12
1.9 PTP1B as an exciting target for therapeutic discovery .... 14
1.10 PTP1B mechanism of action ....................................... 15
i
1.11 Conclusion ............................................................... 17
1.12 References .............................................................. 18
CHAPTER TWO: SYNTHESIS OF NOVEL 2-AMINOPYRIMIDINE CHLORIDES, SULFONAMIDES AND ITS AMINO ACID ANALOG ........................................................... 28
2.1 General introduction ................................................. 28
2.2 Results and discussion .............................................. 32
2.3
CHAPTER THREE: SYNTHESIS OF ARYL-1,2-EPOXY CARBOXYLATES
3.3 Carboxylates as tyrosine phosphatase inhibitors ........... 68
3.4 Resu
3.6 Conclusion and future directions ................................. 77
Biological activity studies ........................................... 38
2.4 Conclusion ............................................................... 38
2.5 Experimental procedures ........................................... 39
2.5.1 General .................................................. 39
2.6 References .............................................................. 59
AND PHOSPHATES ............................................ 66
3.1 General introduction ................................................. 66
3.2 Phosphonates as tyrosine phosphatase inhibitors .......... 68
lts and discussion .............................................. 69
3.4.1 Synthesis of α-aryl α,β-epoxy phosphonates ........ 69
3.4.2 Synthesis of α-aryl-α,β-epoxy carboxylates .......... 70
3.4.3 Synthesis of α,β-aziridino carboxylates ................ 73
3.5 Biological activity studies ........................................... 76
ii
3.7 Experimental procedures ........................................... 78
3.8 References .............................................................. 98
CHAPTER FOUR: SYNTHESIS OF STANDARD AND CYSTEINE BASED PEPTIDE NUCLEIC ACID (CPNA) MONOMERS AND OLIGOMERS ................................................... 102
4.1 General introduction ................................................ 102
4.2 Results and discussion ............................................. 107
4.2.1 Synthesis of standard PNA monomers ................ 107
Synthesis of CPNA monomers ........................... 109 4.2.2
4.5 Conclusion .............................................................. 114
4.6 Experimental procedures .......................................... 114
4.7 References ............................................................. 140
CHAP
About the Author ........................................................... End Page
4.2.3 Synthesis of novel polyether side chain .............. 111
4.3 Solid phase synthesis of PNAs ................................... 112
4.4 Solid phase synthesis results ..................................... 113
TER FIVE: APPENDICES ..................................................... 136
Appendix A:-Selected 1H and 13C NMR spectra ..................... 147
iii
LIST OF FIGURES
Figure 1.1 Oxidative phosphorylation in the biosynthesis of ATP ....... 2
Reversible phosphorylation on protein controlled by Figure 1.2
kinase and phosphatase enzymes ................................. 3
Figure 1.3 Typical amino acid residues for phosphorylation .............. 5
Figure 1.4 Leading causes of death in US (2009) in adults over 25
10
Figure 1.6 Some
Figure 1.7 Activity and selectivity of some prominent inhibitors ...... 13
Figure 1.8 PTP1B mechanism of action ....................................... 16
Figure 1.9 Proposed pyrimidine based phosphatase inhibitors ........ 17
Figure 1.10
Figure 2.1
Figure 2.2
Figure 3.1
years ........................................................................ 8
Figure 1.5 Phosphotyrosine and commonly used pTyr mimetics .....
representative phosphatase inhibitors ................ 11
Proposed epoxy and aziridine based phosphatase
inhibitors ............................................................... 17
Some important drugs with sulfonamide functional
groups .................................................................... 30
Proposed 2-aminopyrimidine derivatives as novel
PTP1B inhibitors ....................................................... 31
Conversion of pyruvate to lactate in respiratory cycle .... 67
iv
Figure 3.2 Some of the phosphatase inhibitors with carboxy
functionality ............................................................ 68
Figure 4.1.a DNA double helix structure-sugar phosphate
backbone-adenine, thymine, guanine, cytosine
Figur
Figure 4.2 Structures of achiral PNA backbone and chiral DNA
backbone ............................................................... 105
Figure 4.3 Proposed structure of cysteine based PNA (CPNA) ........ 106
bases ................................................................. 103
e 4.1.b Purines and pyrimidines of nucleic acids, DNA and
RNA.................................................................... 104
v
LIST OF SCHEMES
Scheme 2.1 Synthesis of 5-ethynyl-2-N-[(4-methoxybenzyl)
(methanesulfonyl)] -2-aminopyrimidine 2.5 ............... 33
Scheme 2.2 Synthesis of symmetrical bipyrimidine
sulfonamide 2.8 .................................................... 34
Scheme 2.4 Synthesis of bipyrimidine sulfonyl chloride .......... 36
Scheme 2.5 Synthesis of N-benzyl-C-chloro-N-(5-iodo-pyrimidin-2-
yl)-methanesulfonamide 2.18 .................................. 37
Scheme 2.6 Synthesis of 3-[2-(Benzyl-chloromethanesulfonyl-
propionic acid 2.24 ................................................. 38
Scheme 3.1 Synthesis of α,β-epoxy phosphonate 3.4 ................... 69
Scheme 3.2 Synthesis of α,β-epoxy phosphonic acid 3.6 ............... 70
m
..... 74
Scheme 2.3 Synthesis of 5-iodo-2-N,N di tert butoxycarbonyl
bipyrimidine 2.10 ................................................... 35
2.15
amino)-pyrimidin-5-yl]-2-tert-butoxycarbonylamino-
Scheme 3.3 Synthesis of α,β-epoxy α-aryl carboxylates 3.9a-i ....... 71
Scheme 3.4 Attempts of synthesis of epoxy carboxylate with
ethanal equivalents .............................................. 72
Scheme 3.5 Synthesis of α,β-aziridino carboxylate 3.14 ...........
Scheme 3.6 Synthesis of N-alkyl α,β-aziridino carboxylate 3.19 ..... 75
vi
Scheme 3.7 Synthesis of N-benzyl aziridine carboxylate 3.20 ........ 76
Scheme 3.8 Synthesis of α,β-diaryl α-cyclopropane
carboxylate 3.22 ................................................... 76
Scheme 4.1 Synthesis of Fmoc protected PNA monomers ............. 108
Scheme 4.2 Synthesis of Boc protected PNA monomers ............... 109
Scheme 4.3 Synthesis of novel cysteine PNA monomers .............. 110
Scheme 4.4 Synthesis of novel polyether side chain .................... 111
vii
LIST OF TABLES
lid phase synthesis of PNAs..................................... 113 Table 4.1 So
viii
LIST OF SYMBOLS AND ABBREVIATIONS
1o = Primary
2o = Secondary
3o = Tertiary
3D = Three dimensional
Å = ångström
AA = Amino acid
Ac = Acetyl
Ar = Aryl
ADP = Adenosine diphosphate
ATP = Adenosine triphosphate
Boc = tert-Butoxycarbonyl
Bn = Benzyl
oC = degrees Celsius
13C = carbon 13
Calcd = calculated
Cbz = Benzyloxycarbonyl
CDI = Carbonyldiimidazole
Cys = Cysteine
Bt = Benzotriazole
ix
DBU = 1,8-Diazabicyclo[5.4.0]undec-7-ene
DCC = 1,3-Dicyclohexylcarbodiimide
DCM = Dichloromethane
DIBAL = Diisobutyl aluminum hydride
DIEA = Diisopropylethylamine
DMAP = 4-Dimethylaminopyridine
DME or 1,2-DME = Dimethoxyethane
DMF = N,N-Dimethylformamide
DMS = Dimethylsulfide
DMSO = Dimethylsulfoxide
dsDNA = double strand deoxyribonucleic acid
DSPs = Dual specific phosphatases
ESI MS= Electrospray ionization mass spectrometry
Fmoc = 9-Fluorenylmethoxycarbonyl
HATU = O-(7 ium
hexaflurophosphate
HIV = Human immunodeficiency virus
HOBt = 1-Hydroxybenzotriazole
HRMS = High resolution mass spectrum
IRs = Insulin receptors
IRSs = Insulin receptor substrates
LCMS = Liquid chromatography – mass spectrometry
-azabenzotriazol-1yl)-1,1,3,3,tetramethyluron
x
NBS = N-Bromosuccinamide
Methylmorpholine
ance
nosulfate
nesulfonyl azide
nzyl
acid
sphatases
sine kinases
hosphatase 1B
sphatases
p-Toluenesulfonic acid
g tyrosine phosphatase 2
de
NMM = N-
NMR = Nuclear magnetic reson
Oxone® = Potassium peroxymo
ρABSA = 4-Acetamidobenze
Ph = Phenyl
PMB = 4-methoxybe
PNA = Peptide nucleic
PPs = Protein pho
PTKs = Protein tyro
PTP1B = Protein tyrosine p
PTPs = Protein tyrosine pho
PTSA/p-TsOH =
Py = Pyridine
R = Alkyl
RNA = Ribonucleic acid
SAR = Structure activity relationship
SHP-2 = SH2 domain-containin
TBAF = Tetrabutylammonium fluori
TBAI = Tetrabutylammonium iodide
TEA = Triethylamine
TES = Triethylsilane
xi
xii
nyl /Triflate
cid
Tf = Trifluoromethanesulpho
TFA = Trifluoroacetic acid
TFMSA = Trifluoromethanesulfonic a
TMEDA = N,N,N',N'-Tetramethylethylenediamine
TMSCl = Chlorotrimethylsilane
ABSTRACT
Protein phosphorylation is a post translational modification of
proteins in which a serine, a threonine or a tyrosine residue is
phosphorylated by an enzyme, kinase. Phosphorylation of proteins is a
reversible and very important regulatory mechanism that occurs in
both prokaryotes and eukaryotes. Phosphorylation turns many protein
enzymes on and off, preventing or causing many diseases such as
diabetes, cancer and rheumatoid arthritis. The phosphorylation on
tyrosine residues of proteins is essential for transmission of signals for
cell growth, proliferation and differentiation. Protein tyrosine
phosphatases (PTPs) in concert with protein tyrosine kinases (PTKs)
regulate many signal transduction pathways by controlling the degree
of phosphorylation of tyrosine residues within the protein. While the
roles and mechanisms of protein tyrosine kinases are well
documented, our present understanding of protein tyrosine
phosphatases is very limited. In this regard we still have much more to
learn about PTPs. Here we propose the design and synthesis of novel
protein tyrosine phosphatase mimetics and their activity against
tyrosine phosphatases. Chapter two describes the synthesis of 2-
aminopyrimidine chlorides, sulfonamides and the sequence of
reactions to make its amino acid analog. Chapter three describes the
synthesis of α-aryl, α,β-epoxy carboxylates, phosphonates and their
xiii
xiv
biological activity against tyrosine phosphatases. These compounds
could be very helpful in significantly improving the current
understandings about the roles and mechanisms of the PTPs. These
proposed tyrosine phosphatase inhibitors are believed to work
effectively in treating the diseases by modulating the phosphorylation
in signal transductions pathways. Chapter four describes the design
and the synthesis of Peptide Nucleic Acids (PNAs) both standard as
well as hybrid PNAs with novel cysteine based monomers that are
aimed to increase the cellular uptake by introducing positively charged
or amphipathic species attached to cysteine thiol functional group.
CHAPTER ONE
PROTEIN PHOSPHATASE INHIBITORS
1.1 General introduction to phosphorylation
Phosphorylation is the addition of an inorganic phosphate group
to an organic molecule or a protein. It creates adenosine triphosphate
(ATP), an energy storing molecule, from adenosine diphosphate (ADP)
by the addition of an inorganic phosphate group in living cells (Figure
1.1). ATP, also known as the energy currency of the cell, is the form of
energy needed to sustain our cells and thereby sustain every living
organism.1,2 Protein phosphorylation in particular is an important
cellular process of living organisms both prokaryotes and eukaryotes.
It has immense potential in understanding and curing some of
challenging diseases and this is evident from the thousands of
research articles being published on this topic every year in chemistry
and biochemistry disciplines. Phosphate groups on a protein were first
identified by Phoebus A. Levene in vitellin in 19063 and this
observation was confirmed by Fritz Lipmann in 1933 in casein.4
Enzymatic protein phosphorylation was then described for the first
time by Eugene P. Kennedy in 1954.5
1
Figure 1.1. Oxidative phosphorylation in the biosynthesis of ATP
Phosphorylation plays a crucial role in many cellular processes as
in biological thermodynamics, enzyme activation, enzyme inhibition,
protein-protein recognition, and protein degradation. Phosphorylation
of ATPase during the transport of the metal ions Na+ and K+ across the
cell membrane in osmoregulating to maintain homeostasis of the
body’s water content,6 phosphorylation of the enzyme GSK-3 by
protein kinase B (AKT) as part of the insulin signaling pathway,7
phosphorylation of src tyrosine kinase by Csk,8 phosphorylation of
NADPH oxidase9 are a few examples which indicate the significance of
phosphorylation. It’s been recognized that phosphorylation of some
proteins causes them to be degraded by the ATP-dependent
ubiquitin/proteaosome pathway. These target proteins will become
substrates for particular E3 ubiquitin ligases only when they are
phosphorylated.10
2
1.2 Protein phosphorylation
Enzymatic protein phosphorylation is a reversible process in
most living organisms on the planet. Kinases catalyze the
phosphorylation and phosphatases catalyze dephosphorylation.
Reversible protein phosphorylation is the basis for the regulation of
diverse cellular processes that include cellular metabolism,
contractility, transport, cell division, differentiation and development,
learning and memory.11-13
Figure 1.2 Reversible phosphorylation on protein controlled by kinase and phosphatase enzymes
The extent of phosphorylation and dephosphorylation turn many
enzymes on and off by changing the conformation of the
corresponding enzymes and the active sites of the receptors. The usual
3
sites of phosphorylation on the proteins are the amino acid moieties
which have hydroxyl functional groups as in serine, threonine and
tyrosine. In primitive organisms the sites of the phosphorylation also
include histidine, arginine, cysteine and lysine (Figure 1.3).14
Phosphorylation increases the hydrophilicity of the particular group
and can sometimes occur on the side chains of the amino acids that
are otherwise in hydrophobic protein patches such as occurs on the
p53 tumor suppression protein.15 The sensitive cells on the retina get
activated upon phosphorylation and then they process the incoming
light in the signal transduction. The extraordinary diversity of both
kinases (over 500) and phosphatases (over 100) that have been
identified in the human genome itself speaks to the essential roles
these proteins play in daily biological processes taking place in the
body.16
Figure 1.3 Typical amino acid residues for phosphorylation (second row amino acids are phosphorylation sites in primitive organisms)17
4
Phosphorylated proteins can be dephosphorylated by either
specific phosphatases like serine/threonine specific or tyrosine specific
phosphatases or non specific phosphatases like alkaline phosphatases.
1.3. Occurrence of protein phosphorylation
Protein phosphorylation can occur on multiple sites in the same
protein at any given time and it is estimated that approximately 30%
of the 10000 proteins in a typical mammalian cell are thought to be
phosphorylated at any given time.18 This suggests that many cellular
functions could be artificially altered if one could control the activity of
kinases or phosphatases or both. This led to enormous interest in
identifying and studying small molecules both peptidic and non-
peptidic, on specific kinases and phosphatases. Since the
phosphorylation is crucial for stabilizing a particular conformation of a
protein in order to act as an enzyme or as signal transduction element
it is very important to know the state of phosphorylation of the protein
to understand the structure and function of the same.
As mentioned earlier the major sites of phosphorylation in
advanced organisms, including human beings, are serine, threonine
and tyrosine. Tyrosine sites are very rare sites for phosphorylation;
however the corresponding proteins are easily purified by antibodies
and hence well understood.19
5
1.4 Protein phosphatases and their importance
Protein phosphatases (PPs) are an important sub family of
enzymes that remove phosphate groups from proteins and work
exactly opposite to the function of protein kinases which phosphorylate
proteins and together these opposing enzymes maintain the
equilibrium at right levels. Protein phosphatases were initially classified
into families based on their dephosphorylation sites whether they
dephosphorylate Threonine/Serine residues, Tyrosine residues (PTPs)
or both of them (dual specific phosphatases-DSPs). But now it has
been discovered that certain Ser/Thr specific enzymes can
dephosphorylate Tyr, and many of the enzymes in the dual specific
family based on the sequence can selectively function on Tyr, Ser, and
Thr, RNA or phosphoinositides.
Some important phosphatases include CD45, PTP1B, PTP1N,
TCPTP, SHP-2, LAR, PP2C, Ppz1p, Ppz2p. CD45 is present in human
hematopoietic cells and its mutation may lead to dysfunction of B
lymphocytes and low T cell production which can lead to
immunodeficiency. PTP1B is a negative regulator of insulin and is
considered as a potential therapeutic target to treat type-II diabetes.
TC-PTP is prominent in hematopoietic cell types and plays critical role
in bone marrow maturation.20-22
6
1.5 Protein tyrosine phosphatases (PTPs)
Protein tyrosine phosphatases (PTPs) are an important super
family of enzymes which remove phosphate groups from
phosphorylated tyrosine residues on proteins and regulate many signal
transduction pathways, including growth initiation, propagation and
termination, by regulating the extent of phosphorylation by working in
association with the kinases in reciprocal directions. Defects in PTPase
activity can lead to aberrations in the phosphorylation of tyrosine,
eventually leading to protein malfunction, which contributes to the
many human diseases like cancer, diabetes, obesity and rheumatoid
arthritis. In recent days, PTPases have gained importance as the drug
discovery target because of its critical role in bioprocesses. There are
many classes of molecules being synthesized and studied for their
activity on phosphatases some of which are shown in the Figure 1.6.23
Obesity is the second largest cause of preventable deaths after
smoking in the United States. Obese people are more prone to develop
associated diseases than the people with normal weight like diabetes,
heart diseases, strokes, high blood pressure, cancer and obstructive
sleep apnea. 0.6 Million people are estimated to die because of cancer
in the US alone which is alarming (Figure 1.4)
7
Figure 1.4. Leading causes of death in US (2009) in adults over 25 years24
Type II diabetes alone afflicts over 200 million people worldwide
and many more are unaware they are at high risk. The number of
people diagnosed is expected to grow steadily over the next several
years. Type II diabetes (non insulin-dependent diabetes mellitus
(NIDDM) or T2DM), is characterized by a resistance to insulin which
can be due to the ignorance of the body to the insulin which is
necessary for the body to be able to process glucose for the required
energy needs or because of inadequate beta cell activity and it
accounts for 90% of all diabetic patients. Increased fatty acid oxidation
contributes greatly to hyperglycemia by formation of high levels of
acetyl-coenzyme A, ATP, and NADH, which increase gluconeogenesis
and thus hepatic glucose production. Type I diabetes, insulin-
dependent diabetes mellitus (IDDM or T1DM), results from a lack of
production or under production of insulin. When we eat food, the body
8
processes all of the sugars and starches into glucose, which is the
energy currency of the cell. Insulin signals cells to take the sugar from
blood. In diabetic conditions, glucose builds up in the blood instead of
going into cells.25
1.6 Phosphotyrosine mimetics
The discovery of a PTP1B knockout as a non lethal mutation with
increased insulin sensitivity by Elechbly et al has lead to the realization
among the scientific community that the inhibition of protein tyrosine
phosphatases might be a valuable research tool for insulin biology
research.26 It could also be a possible source of drug development for
treating major diseases like diabetes and obesity. The research in this
field can lead to treatments of infectious agents, neurological
disorders, autoimmune and certain cancers. To understand the
function of phosphatases and in particular tyrosine phosphatases
various molecules were developed and studied to find the exact
mechanism of action of various phosphatases. Some of the well
studied mimetics are shown in Figure 1.5.
9
Figure 1.5 Phosphotyrosine and commonly used pTyr mimetics27
1.7 Phosphatase inhibitors
The initial findings of the PTP1B knock out experiments lead to
the tremendous interests in both academia and industry to develop
and study peptidic and non-peptidic molecules as the potential
inhibitors of the protein phosphatases. The first crystal structure for
PTP1B has been solved by Tonks et al in 1994.28 There are currently
around 20 phosphatases with crystal structures that been solved. The
crystal structure findings are valuable for the design of new inhibitors
as they showed the secondary sites, some times called satellite sites,
clearly in addition to the primary active sites. It’s been common to
design molecules to interact with the secondary sites in addition to the
active site increasing the specificity of the inhibitor to target desired
10
phosphatase enzymes. Some representative molecules are shown in
the Figure 1.6.
Figure 1.6 Some representative phosphatase inhibitors29-30
11
1.8 Challenges in designing a potent & specific phosphatase inhibitor
The evolution of all of the phosphatases is believed to be from
the same ancestor according to some of the biologists and it explains
the very close similarities among phosphatases.31 All protein tyrosine
phosphatases carry a highly conserved active site signature motif, [the
(H/V)CX5R(S/T) amino acid sequence], which adopts a unique loop
structure and employ a common catalytic mechanism. They have
similar core structure made of a central parallel beta sheet with
flanking alpha helices containing a β-loop and α-loop surrounding
signature motif.32 The functions and specificity of these proteins are
attributed to the regulatory domains and subunits. Because of the
close similarities among the PTPs, it is known to be very difficult to
selectively target specific PTPase and PTP1B in particular. For example
PTP1B is closely related to TC-PTP and almost 80% of the sequence is
similar in both of these phosphatases. This makes it very challenging
to control selectivity. Some of the best known phosphatase inhibitors
are shown and their biological activities and selectivities are compared
in the Figure 1.7.33
12
Figure 1.7 Activity and selectivity of some prominent inhibitors
In addition to the similarities in structures specific PTPase may
also regulate multiple signaling pathways and similarly one signaling
pathway can be regulated by multiple PTPases.34
In human beings proteins are predominantly phosphorylated on
three residues Ser, Thr and Tyr residues, with each accounting for
approximately 86%, 12% and 2% respectively. Human DNA encodes
13
~ 520 protein kinases, with nearly 428 known or predicted to
phosphorylate Ser and Thr residues and 90 belonging to the tyrosine
kinase family. In contrast there are only ~ 107 human phosphatases
and only around 81 are predicted to be active protein phosphatases
(based on human genome findings).35-38
In addition, since the active site of PTP1B is highly hydrophilic, it
remains a challenge to identify inhibitors with both excellent in vitro
potency and drug-like physiochemical properties which would lead to
good in vivo activities. Major breakthroughs in designing potent
inhibitors have to address the following two important issues 1) Many
PTPs have similar structures and sequences in their active site regions.
This makes it more difficult to design inhibitors that are specific for the
corresponding target. 2) Most of the small molecules that bind with
high affinity in these active sites are hydrophilic leading to poor cell
permeability.39-40
1.9 PTP1B as an exciting target for therapeutic drug discovery
Since the findings of PTP1B as a target for finding new drugs to
the existing problems of obesity and diabetes in biology, many
research groups have come up with different functional units based on
theoretical and experimental results both peptidic and non peptidic
14
molecules. It is worth to mention some of the molecules reached to
clinical trials II and failed to reach further due to efficacy issues.41-42
1.10 PTP1B mechanism of action
The protein tyrosine phosphatase PTP1B became an exciting
target for the treatment of type II diabetes and obesity. The enzyme is
a negative regulator of the insulin signaling pathway acting by
dephosphorylating phosphotyrosine residues in the insulin receptors
(IRs) and insulin receptor substrates (IRSs). To date more than 20
PTPs have been solved for their crystal structures.43 The PTPs are
composed of β-barrels flanked by α-helices. The catalytic site is
located in a groove at the protein surface which is 9 Å deeper for
classical PTPs compared to DSPs which are 6 Å deep. This difference is
responsible for the higher substrate selectivity of the classical PTPs.
Recognition of the substrate peptide sequence by PTP1B and binding of
the phosphotyrosine deep in the catalytic site of the phosphate-binding
loop (P-loop) are mediated by residues 214-221. The WPD loop
consisting of tryptophan, proline and aspartic acid closes down onto
the substrate and thereby positions the thiolate of cysteine 215 for
nucleophilic attack at the electrophilic phosphorous on the
phosphotyrosine. Aspartic acid 181 acts as a general acid catalyst. This
mechanism eventually hydrolyses to give inorganic phosphate group
15
which diffuses from the active site and gets replaced with water
molecule. (Figure 1.7)44-45
Figure 1.8 PTP1B mechanism of action
After carefully looking at current molecules being used as
inhibitors and understanding the PTP1B mechanism of action we herein
propose design and synthesis of molecules which we believe could
work as non covalent46 or covalent inhibitors47 for different
phosphatases paying particular attention to inhibit PTP1B selectively
(Figure 1.8 and Figure 1.9). Pyrimidine chloride 2.24 is particularly
interesting as we will use this molecule to insert into small peptide and
study its effect as it is our belief that this will increase the selectivity
dramatically.48-50
16
Figure 1.9. Proposed pyrimidine based phosphatase inhibitors
Figure 1.10. Proposed epoxy and aziridine based phosphatase inhibitors
1.11 Conclusion
It’s been challenging to discover potent cell permeable and orally
bioavailable PTP1B inhibitors. The highly cationic nature of the active
site and the lack of adjacent hydrophobic binding sites have been
obstacles in developing potent inhibitors. In this regard, we designed
some of the molecules shown in Figures 1.8 and 1.9 as phosphatase
17
inhibitors and synthesized some of them and tested for their activity
against protein tyrosine phosphatases (PTPs) and the activities are
tabulated in the respective chapters.
1.12 References
1. Cohen, P. The origins of protein phosphorylation. Nat. Cell. Biol.
2002, 4 (5), 127-130.
2. Lipmann, F. A.; Levene, P. A. Serinephosphoricacid obtained on
hydrolysis of vitellinic acid. J. Biol. Chem. 1932, 98 (1), 109-
114.
3. Levene, P. A.; Alsberg, C. L. The Cleavage of Products of Vitellin.
J. Biol. Chem. 1906, 2 (1), 127-133. b) Kenner, K. A.;
Anyanwu, E.; Olefsky, J. M.; Kusari, J. Protein tyrosine
phosphatase 1B is a negative regulator of insulin- and insulin-
like growth factor-I-simulated signaling. J. Biol. Chem. 1996,
271, 19810-19816.
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CHAPTER TWO
SYNTHESIS OF NOVEL 2-AMINOPYRIMIDINE CHLORIDES,
SULFONAMIDES AND ITS AMINO ACID ANALOG
2.1 General introduction
Ever since the introduction of synthetic sulfonamide drugs
(popularly known as sulfa drugs) as antibiotics in the early 1930s,
there has been significant attention being paid to the discovery of new
and effective drugs with the sulfonamide functional group. There are
currently over 15,000 sulfonamide derivatives, analogs and related
compounds, which have been synthesized and tested for different
diseases. This studies lead to the discovery of so many useful
sulfonamide medicines for treating urinary, thyroid, heart diseases,
malarial infection and leprosy along with many other diseases.1
Sulfonamide drugs were the first antimicrobial agents
administered in combination with antibiotics for the treatment of
infection. When penicillin and streptomycin first became available they
were often given in combination with sulfonamides in which cases they
became less effective by themselves.2 In some instances they were
used as additives and in some they acted synergistically. In 1932,
German chemist Gerhard Domagk discovered that a dye called
28
Prontosil controlled streptococcal infections in mice for which he later
received the Nobel Prize in 1939 in the field of Physiology and
Medicine.3 Prontosil was also found to control staphylococcal infections
in rabbits without harming animals. Since then, sulfonamides have
been clinically used for decades and have been found to have wide
ranging biological activities including antiviral, antidiabetic
(hypoglycemic), diuretic, and antithyroid activities and also antitumor
agents.4-6 The sulfonamide functional group is present in well
established thiazide diuretics, loop diuretics, sulfonyl ureas, COX-2
inhibitors,7 anti-inflammatory agents, anti-rheumatics, anti-
ulceratives, acetazolamide and many more (Figure 2.1). Folate
synthesis is the important step in the synthesis of bacterial cell wall.
Antibacterial sulfonamides act as competitive inhibitors of the enzyme
dihydropteroate synthetase; an enzyme involved in folate synthesis,
and prevents the formation of cell wall which ultimately prevents the
growth of bacteria. They have been also been used in retroviral
therapy as HIV protease inhibitors recently.8
29
Figure 2.1. Some important drugs with sulfonamide functional groups
The compounds with sulfonamide functional group have became
increasingly popular recently among medicinal chemists in part
because of the ease of deprotonation at physiological pH in the body
which makes them more water soluble, more drug-like properties,
which is a fundamental requirement for any chemical entity to be used
as an effective drug in treating any kind of diseases.9-12
We, keeping in mind the fundamental principles of drug
discovery low toxicity and cellular solubility, based our target
molecules on commercially readily available 2-aminopyrimidine in part
due to its low toxicity towards the living cells and the pKb of the NH
proton in the product sulfonamide.13-14
30
Figure 2.2 Proposed 2-aminopyrimidine derivatives as novel PTP1B inhibitors
We focused our efforts to synthesize potent, yet highly selective
inhibitors for individual members of the large PTPase family of
enzymes paying particular attention to PTP1B. 15-17 In this regard, we
came up with the 2-aminopyrimidine derived molecules shown in the
Figure 2.2. The compound 2.8 is expected to work as a non covalent
inhibitor. The other three compounds expected to work as either
covalent or non covalent inhibitors due to the electrophilic character.18
To increase the specificity of the inhibition for PTP1B we were
interested in making a small peptide library, to obtain structure
activity relationship (SAR) data, containing the amino acid derived
chloride 2.24 shown in Figure 2.2. For this, we will employ solid phase
peptide synthesis methods.19
31
Biological activity is explained briefly at the end of this chapter.
Some of the key synthetic reactions in this chapter are the
Sonogashira cross coupling, introduction of acetylene moiety and the
deprotection of tert-butoxycarbonyl groups from the 1o amine group
employing an environmentally friendly catalyst, Montmorillonite K-10
clay.
2.2 Results and discussion
Professor Jerry Wu at the Moffitt Cancer Center collaborated with
us by studying the biological activity of the compounds synthesized in
this chapter.20 Dr. Wayne Guida’s group supported us with the design
and modeling data for the same. Fenger Zhou’s contribution in scaling
up intermediate 2.5 is also appreciated.
Syntheses of all of the 2-aminopyrimidine derivatives discussed
in this chapter were started from the commercially available 2-
aminopyrimidine. 2-Aminopyrimidine was reacted with
methanesulfonyl chloride in pyridine as solvent to afford 2-
aminopyrimidine sulfonamide 2.1 in good yields.21 Reaction of the
above sulfonamide with molecular iodine in the presence of mercuric
acetate in hot 1,4-dioxane solvent afforded 5-iodo-2-N-
(methanesulfonyl)aminopyrimidine 2.2 in excellent yields.22
32
Sulfonamide 2.2 was protected as the para-methoxybenzyl
sulfonamide 2.3 employing very well established reaction conditions
using para-methoxybenzyl chloride, KI and K2CO3 in DMF at ambient
temperatures in very good yields.23 Pd (0) catalyzed Sonogashira
coupling conditions24 were employed to introduce the desired alkyne
functionality in the molecule using commercially available 2-methyl-3-
butyn-2-ol which gave alkyne-ol 2.4 in excellent yields. The same was
deprotected using NaOH in toluene as solvent at reflux temperatures
to afford intermediate alkyne 2.5 in moderate yields.25 (Scheme 2.1)
Scheme 2.1 Synthesis of 5-ethynyl-2-N-[(4-methoxybenzyl) (methanesulfonyl)]-2-aminopyrimidine 2.5
33
Alkyne 2.5 and iodide 2.3 were subjected to the Sonogashira
coupling conditions mentioned earlier to get the coupled product, the
symmetrical alkyne 2.6, in very good yields which was subsequently
hydrogenated over hydrogen gas using 10% (w/w) Pd over carbon26
as catalyst in EtOH: EtOAc: DCM (12:12:1) solvent system to afford
the symmetrical bipyrimidine 2.7 in quantitative yields. The para-
methoxy benzylamine 2.7 was deprotected to give the bipyrimidine 2.8
in good yields.27 (Scheme 2.2)
Scheme 2.2 Synthesis of symmetrical bipyrimidine sulfonamide 2.8
34
Next we needed 5-iodo-2-N-(di tert-butoxycarbonyl)pyrimidine
as an intermediate to synthesize the chloro analogue 2.15.
Commercially available 2-aminopyrimidine was subjected to aromatic
electrophilic substitution reaction with molecular iodine and mercuric
acetate in hot 1, 4-dioxane and water (3:1) at 70 oC to afford 5-iodo-
2-aminopyrimidine 2.9 in very good yields. The obtained amine was
protected as a di tert-butyl dicarbamate 2.10 using di tert-butyl
dicarbonate and DMAP in DMF at ambient temperatures.28 (Scheme
2.3)
Scheme 2.3 Synthesis of 5-iodo-2-N,N-di tert butoxycarbonyl bipyrimidine 2.10
Alkyne 2.5 and iodide 2.10 were coupled under Sonogashira
coupling conditions as previously mentioned to give alkyne 2.11 in
good yields, which on hydrogenation gave bipyrimidine 2.12 in
excellent yields. The product was heated to reflux in acetonitrile with
montmorillonite K-10 clay to deprotect tert-butoxycarbonyl groups and
obtain quantitative yields of 2.13 without needing further purification.
Sulfonation with chloromethanesulfonyl chloride followed by the
35
deprotection of PMB group gave the desired chloride 2.15 in 34%
yields for two steps29 (Scheme 2.4).
Scheme 2.4 Synthesis of bipyrimidine sulfonyl chloride 2.15
Suitably protected 5-iodo-pyrimidine chloride 2.18 was
synthesized from 2-amino pyrimidine using the reaction conditions
mentioned earlier. The PMB group was replaced by benzyl so as to
make it compatible for the selective deprotection of the methyl ester30
of amino acid derivative to get amine protected amino acid 2.24 ready
to use in solid phase synthesis to make small peptides and convenient
deprotection of benzyl group applying Pd catalyzed hydrogenation
conditions. (Scheme 2.5)
36
Scheme 2.5 Synthesis of N-Benzyl-N-(5-iodo-pyrimidin-2-yl)-chloromethanesulfonamide 2.18
Readily available L-Serine was esterified using thionyl chloride to
methyl ester 2.19 and the resulting HCl salt was reacted with di-tert
butyl dicarbonate in the presence of potassium carbonate in THF:H2O
(3:1) solvent to yield N-(tert-butoxycarbonyl)-L-serine methyl ester
2.20 in quantitative yields.31 Tosylation with p-toluenesulfonyl chloride
and subsequent iodination with NaI in acetone afforded the N-(tert-
butoxycarbonyl)-β-iodoalanine methyl ester 2.22 in excellent yields32
which can be further coupled to iodide 2.18 using Pd2dba3 as the
catalyst to get chloro sulfonamide 2.23.33 Selective hydrolysis of the
methyl ester using sodium hydroxide in tetrahydrofuran can result in
the orthogonally protected amino acid 2.24 which can be readily put
into small peptides to increase the selectivity of the inhibition of
selective phosphatase over closely related phosphatases. We will
employ solid phase peptide synthesis to prepare a library of small
peptides incorporating (Scheme 2.6) amino acid 2.24.
37
Scheme 2.6 Synthesis of 3-[2-(Benzyl-chloromethanesulfonyl-
amino)-pyrimidin-5-yl]-2-tert-butoxycarbonylamino-propionic acid 2.24
2.3 Biological activity studies
The compounds synthesized in this chapter 2.8, 2.15 and 2.16
were tested for their activity against SHP-2 and PTP1B but showed no
significant activity.
2.4 Conclusion
Novel 2-aminopyrimidine chlorides and sulfonamides were
synthesized and their application to synthesize amino acid analog 2.24
with tert-butoxycarbonyl as protecting group for solid phase synthesis
of small peptides were proposed and the synthesis was partially
applied to achieve novel peptides as potential noncovalent inhibitors
for tyrosine phosphatases. The final two steps shown in the scheme
2.6 can be easily performed by utilizing the palladium catalyzed
38
coupling conditions and the subsequent hydrolysis of methyl ester to
get 2.24.
2.5 Experimental procedures
2.5.1 General 1H-NMR and 13C-NMR spectra were recorder on a Brucker 250
MHz and the Varian 400 MHz spectrometer in CDCl3, Methanol-d4 and
DMSO-d6 with TMS as the standard. Chemical shifts are reported in
ppm, spin multiplicities are indicated by s (singlet), d (doublet), t
(triplet), q (quartet), p (pentet), m (multiplet), dd (doublet of doublet)
and bs (broad singlet). Thin-Layer chromatography (TLC) was
performed on glass plates coated with 0.25 mm thickness of silica-gel.
All solvents were dried and distilled prior to use and organic solvent
extracts were dried over Na2SO4. Mass measurements were carried out
on ESI LC MS system (Agilient Technologies) and High Resolution Mass
measurements were done on LC MSD TOF system (Agilient
Technologies). MALDI-TOF measurements were recorded on Autoflex
(BRUKER) Melting points were recorded using Melt-Temp
(Electrothermal) instrument and were uncorrected.
39
2-N(methanesulfonyl)-aminopyrimidine (2.1):- To a 250 mL two
necked round bottomed flask equipped with nitrogen inlet was charged
2-aminopyrimidine (32 g, 0.34 mol) followed by the addition of
pyridine (128 mL) under positive pressure of nitrogen. The mixture
was brought to 0 oC using an ice bath. A solution of methanesulfonyl
chloride (72.1 g, 0.63 mol) in pyridine (96 mL) was added over a
period of 10 minutes after which the reaction was brought to ambient
temperatures in one hour. The reaction was stirred for another 10
hours before concentrating under reduced pressure. Residual pyridine
was removed azeotropically by evaporating with methanol (3×40 mL)
before purifying the crude by recrystallization in methanol to get pure
product 2-N-(methanesulfonyl)-aminopyrimidine 2.1 as an off-white
solid. (38.3 g, 65.8%). 1H NMR (DPX 250 MHz, CDCl3) δ 11.32 (bs,
1H, NH), 8.64 (d, J=5.0, 2H), 7.15 (t, J=5.0), 3.38 (s, 3H, CH3); 13C
NMR (DPX 250 MHz, CDCl3) δ 158.54, 157.54, 115.72, 41.25; LCMS
(ESI) m/z calcd for C5H7N3O2S 173.029 found 174.0 [M+H]+, mp
256.3 oC.
40
5-iodo-2-N (methanesulfonyl) amino pyrimidine (2.2):- To a 1L
three necked flask 2-N-(methanesulfonyl)-aminopyrimidine 2.1 (13.0
g, 75.0 mmol) and glacial acetic acid (400 mL) were charged. The
heterogeneous mixture was heated to 120 oC to dissolve all the solids
which resulted in the light brown solution. Iodine (20.0 g, 78.8 mmol)
was charged to the flask in one portion before cooling to room
temperature at which Hg(OAc)2 was charged in one portion. After 5
minutes of stirring at room temperature, the reaction was heated to
120 oC for an hour. Reaction was monitored by thin layer
chromatography. (The disappearance of iodine color indicates the
completion of the reaction). The reaction mixture was carefully poured
into 15% KI solution (975 mL) and stirred for another 30 minutes.
Crude product was collected by filtration and the same was
recrystallized from MeOH to get pure compound, 5-iodo-2-N-
(methanesulfonyl) aminopyrimidine 2.2, as an off-white solid. (19.28
g, 85.8%) 1H NMR (DPX 250 MHz, CDCl3) δ 8.77 (s, 2H), 7.26 (s, 1H),
3.44 (s, 3H); 13C NMR (DPX 250 MHz, DMSO-d6) δ 163.43, 156.19,
41
84.78, 41.12; LCMS (ESI) m/z calcd for C5H6IN3O2S 299.0895 found
299.9 [M+H]+, mp 263.3 oC.
5-iodo-2-N [(methanesulfonyl), (4-methoxybenzyl)] amino
pyrimidine (2.3): To a stirred solution of the iodide 2.2 (2.0 g, 6.69
mmol), KI (0.11 g, 0.67 mmol) and potassium carbonate (1.85 g,
13.38 mmol) in 50 mL of dry DMF under nitrogen atmosphere was
added p-methoxybenzyl chloride at room temperature. Upon
completion of the reaction solvent was evaporated under vacuo and
the residue was dissolved in ethyl acetate (80 mL) and the same was
washed with water (2х15 mL) and brine (3х15 mL), successively.
Organic layer was dried over anhydrous sodium sulfate and the solvent
was evaporated under vacuo. The crude was subjected to the flash
chromatography on silica gel using EtOAc/Hexane (2:8) as eluent to
give pure compound 2.3 as a colorless liquid (2.16 g, 77.14%). 1H
NMR (DPX 250 MHz, CDCl3) δ 8.64 (s, 1H), 7.25 (d, 2H, J=7.50), 6.74
(d, 2H, J=7.50), 5.22 (s, 2H), 3.70(s, 3H), 3.26 (s, 3H); 13C NMR
42
(DPX 250 MHz, CDCl3) δ 162.95, 159.12, 157.54, 129.61, 129.07,
113.89, 84.03, 55.25, 48.83, 42.91; HRMS (ESI) m/z calcd for
C13H14IN3O3S 418.980 found 419.9 [M+H]+, mp 107.5 oC.
5-[(2-hydroxy), (2-methyl)] butynyl-2-N [(methanesulfonyl),
(4-methoxynenzyl)] amino pyrimidine (2.4): To a three necked
round bottomed flask were added the iodide 2.3 (1.0 g, 2.38 mmol),
alkyne (0.4 g, 4.77 mmol), CuI (22.7 mg, 0.12 mmol), PPh3 (62.5 mg,
0.24 mmol), Pd (PPh3)4 (27.5 mg, 0.024 mmol), TEA (2.0 mL) followed
by charging with 25 mL of dry acetonitrile. The solution was brought
to 60 oC. Upon completion of the reaction, in 10 hours, solvent was
evaporated under vacuo. The crude was subjected to flash
chromatography on silica gel using EtOAc/Hexane (2:8) as eluent to
give pure compound 2.4 as a yellow fluffy solid (0.82 g, 92%). 1H NMR
(DPX 250 MHz, CDCl3) δ 8.58 (s, 2H), 7.33 (d, 2H, J=7.50 Hz), 6.81
(d, 2H, J=7.50 Hz), 5.34 (s, 2H), 3.37 (s, 3H), 3.34 (s, 3H), 1.65 (bs,
1H), 1.62 (s, 6H); 13C NMR (DPX 250 MHz, CDCl3) δ 159.78, 159.09,
43
157.07, 129.62, 129.18, 113.86, 112.91, 99.48, 75.40, 65.66, 55.24,
48.71, 42.97, 31.32; LCMS (ESI) m/z calcd for C18H21N3O4S 375.1252,
found, 376.1 [M+H]+.
N-(5-Ethynyl-pyrimidin-2-yl)-N-(4-methoxy-benzyl)-
methanesulfonamide (2.5):- The alkyne-ol 2.4 (0.80 g, 2.13 mmol)
and sodium hydroxide (0.22 g, 5.50 mmol) were taken in toluene (50
mL) and the solution was heated to reflux for 10 hours. Upon
completion of the reaction by TLC the solvent was evaporated under
reduced pressure and the residue was extracted into EtOAc. The
organic layer was further washed with brine solution and dried over
anhydrous sodium sulfate. The solvent was removed under reduced
pressure and the crude obtained was purified by flash chromatography
to get pure acetylene 2.5 as a colorless solid (0.33 g, 49 %). 1H NMR
(DPX 250 MHz, CDCl3) δ 8.57 (s, 2H), 7.26 (d, 2H, J=10.0), 6.74 (d,
2H, J=10.0), 5.26 (s, 2H), 3.69 (s, 3H), 3.28 (s, 3H); 13C NMR (DPX
250 MHz, CDCl3) δ 160.37, 159.13, 157.37, 129.61, 129.13, 113.89,
44
112.29, 83.08, 66.17, 55.24, 48.72, 43.00; HRMS (ESI) m/z calcd for
C15H15N3O3S 317.08, found 318.0 [M+H]+, mp 113.1 oC.
N-(5-{2-[Methanesulfonyl-(4-methoxy-benzyl)-amino]-
pyrimidin-5-ylethynyl}-pyrimidin-2-yl)-N-(4-methoxy-benzyl)-
methanesulfonamide (2.6):- To a three necked round bottomed
flask were added aryl iodide 2.3 (0.26 g, 0.63 mmol), alkyne 2.5 (0.20
g, 0.63 mmol), CuI (5.9 mg, 0.031 mmol), PPh3 (16.5 mg, 0.063
mmol), Pd(PPh3)4 (7.2 mg, 0.006 mmol) and TEA (2 mL) followed by
25 mL of dry acetonitrile. The solution was brought to 60 oC. Upon
completion of the reaction, in 10 hours, the solvent was evaporated
under vacuo. The crude was subjected to flash chromatography on
silica gel using EtOAc/Hexane (2:8) as eluent to give pure symmetrical
alkyne 2.6 as a yellow fluffy solid (0.28 g, 74 %). 1H NMR (DPX 250
MHz, CDCl3) δ 8.63 (s, 4H), 7.29 (d, 4H, J=10.0), 6.76 (d, 4H,
J=10.0), 5.30 (s, 4H), 3.71 (s, 6H), 3.37 (s, 6H); 13C NMR (DPX 250
45
MHz, CDCl3) δ 159.75, 159.15, 157.38, 129.67, 129.05, 113.89,
112.38, 87.99, 55.26, 48.76, 43.03; HRMS (ESI) m/z calcd for
C28H28N6O6S2 608.1512, found 609.1592 [M+H]+, mp 212.4 oC.
N-[5-(2-{2-[Methanesulfonyl-(4-methoxy-benzyl)-amino]-
pyrimidin-5-yl}-ethyl)-pyrimidin-2-yl]-N-(4-methoxy-benzyl)-
methanesulfonamide (2.7):- To a solution of alkyne 2.6 (0.27 g,
0.44 mmol) in ethanol/EtOAc/DCM (12:12:1) (25 mL), 10% (w/w)
palladium on carbon (0.1 g) was added carefully. The reaction was
hydrogenated over H2 gas (45 psi) for 12 hrs. The resulting solution
was filtered carefully over Celite and the Celite cake was washed with
ethyl acetate (25 mL) to get most of the compound off of it. The
organic filtrate was subjected to rotary evaporation to yield pure
46
product 2.7 needing no further purification (~ quant %). HRMS (ESI)
m/z calcd for C28H32N6O6S2 612.1824 found 613.1929 [M+H]+.
N-{5-[2-(2-Methanesulfonylamino-pyrimidin-5-yl)-ethyl]-
pyrimidin-2-yl}-methanesulfonamide (2.8):- Compound 2.7 was
taken in round bottomed flask and cooled to 0 oC in an ice bath
followed by the addition of neat TFA (5 mL). The resulting solution was
stirred at room temperature for 2 hours. Upon completion of the
reaction, the solvent was removed under reduced pressure and the
residue obtained was dissolved in DCM and subjected to rotary
evaporation to azeotropically remove the residual trifluoroacetic acid
(3×15 mL). The crude product obtained was precipitated using diethyl
ether to get pure sulfonamide 2.8 as an off-white solid in moderate
yields (0.57 g, 35 %). 1H NMR (DPX 250 MHz, CDCl3) δ 11.44 (bs, 2H),
47
8.5 (s, 4H), 3.36 (s, 6H), 2.50 (s, 4H) , HRMS (ESI) m/z calcd for
C12H16N6O4S2 372.0674 found 373.0751 [M+H]+.
5-iodo-2-amino pyrimidine (2.9):- A solution of 1.02 g (10.72
mmol) of 2-aminopyrimidine in 12 mL of water was treated with 1.36
g (4.26 mmol) of mercuric acetate and the mixture was stirred for two
minutes on the steam-bath. The initially formed yellow precipitate
quickly turned to a thick white slurry which was treated with a solution
of 1.63 g of molecular iodine, I2, (6.44 mmol) in 12 mL of hot dioxane
at 50 oC. All of the iodine reacted during 30 minutes of stirring during
which time considerable evaporation was observed. The thick slurry
was poured into several volumes of 15% potassium iodide solution and
washed on the filter with fresh iodine solution until white.
Recrystallization from absolute methanol gave pure compound, 2-
amino-5-iodopyrimidine 2.9 as an off-white solid (2.07 g, 87.3 %). 1H
NMR (DPX 250 MHz, DMSO-d6) δ 8.35 (s, 2H), 6.83 (s, 2H); 13C NMR
(DPX 250 MHz, DMSO-d6) δ 172.04, 162.53, 98.25; HRMS (ESI) m/z
calcd for C4H4IN3 220.9450, found [M+H] + 221.9517, mp 222.9 oC.
48
Di-tert-butyl (5-iodopyrimidin-2-yl)dicarbamate (2.10): To a
stirred solution of 5-iodo-2-aminopyrimidine 2.9 (0.50 g, 2.3 mmol) in
DMF (15 mL) were added di-tert-butyl dicarbonate (1.15 g, 5.27
mmol) and 4-dimethylaminopyridine (0.025 g, 0.11 mmol) at room
temperature. Progress of the reaction was monitored by thin layer
chromatography using EtOAc/Hexane (50:50) as mobile phase on
silica coated TLC plates. Upon completion of the reaction, DMF was
evaporated under reduced pressure. Crude product obtained was
subjected to flash chromatography on silica gel using EtOAc/Hexane
(50:50) as eluent to give a pure iodide 2.10 as a colorless solid
(0.652g, 68.4 %). 1H NMR (DPX 250 MHz, CDCl3) δ 8.84 (s, 2H), 1.40
(s, 18H); 13C NMR (DPX 250 MHz, CDCl3) δ 164.09, 157.46, 150.40,
89.83, 83.91, 27.85; LCMS (ESI) m/z calcd for C14H20IN3O4, 421.049
found 444.0, [M+H]+, mp 146.5 oC.
49
(5-{2-[Methanesulfonyl-(4-methoxy-benzyl)-amino]-
pyrimidin-5-ylethynyl}-pyrimidin-2-yl)-di-carbamic acid di-
tert-butyl ester (2.11):- To a three necked round bottomed flask
were added alkyne 2.5 (0.500 g, 1.57 mmol), iodide 2.10 (0.69 g, 1.6
mmol), CuI (0.015 g, 0.078 mmol), PPh3 (0.041 g, 0.15 mmol),
Pd(PPh3)4 (0.018 g, 0.015 mmol) and TEA (2 mL) followed by 30 mL of
dry acetonitrile. The solution was brought to 60 oC. Upon completion
of the reaction, in 10 hours, the solvent was evaporated under vacuo.
The crude was subjected to the flash chromatography on silica gel
using EtOAc/Hexane (2:8) as eluent to give pure alkyne 2.11 as a
white solid (0.78 g, 82 %). 1H NMR (DPX 250 MHz, CDCl3) δ 3.59 (s,
3H), 3.19 (s, 3H), 1.30 (s, 18H); HRMS (ESI) m/z calcd for
C29H34N6O7S 610.2209 found 633.2125 [M+Na]+, mp 83.2 oC.
50
N-{5-[2-(2-Amino-pyrimidin-5-yl)-ethyl]-pyrimidin-2-yl}-N-(4-
methoxy-benzyl)-methanesulfonamide (2.13):- To a solution of
compound 2.11 (0.295 g, 0.48 mmol) in ethanol was added 10 wt%
Pd/C catalyst and the resultant heterogeneous solution is subjected to
hydrogenation as described in the synthesis of 2.7 except using
ethanol as solvent. The obtained product 2.12 was taken in acetonitrile
without further purification and Montmorillonite K-10 clay (0.3 g) was
added carefully. The reaction was refluxed at 82 oC overnight. The
resulting solution was filtered carefully over Celite and the Celite cake
was washed with ethyl acetate (25 mL). The organic filtrate was
subjected to rotary evaporation to obtain pure product 2.13 as a white
solid, needing no further purification, in near quantitative yields. 1H
NMR (DPX 250 MHz, CDCl3) δ 8.31 (s, 2H), 8.04 (s, 2H), 7.26 (d, 2H,
J=7.50), 6.74 (d, 2H, J=7.50), 5.25 (s, 2H), 5.03 (s, 2H), 3.70 (s,
3H), 3.27 (s, 3H), 2.71 (m, 4H).
51
N-{5-[2-(2-methanesulfonylamino-pyrimidin-5-yl)-ethyl]-
pyrimidin-2-yl}-chloromethanesulfonamide (2.15): To a stirred
solution of aryl amine 2.13 (0.16 g, 0.38 mmol) in pyridine (15 mL) at
room temperature was added to chloromethanesulfonyl chloride
dropwise over a period of 10 minutes. Progress of the reaction was
monitored by thin layer chromatography using MeOH/EtOAc (2:8) as
eluent. Upon completion of the reaction solvent was removed under
reduced pressure to give crude compound which on flash
chromatography gave pure product 2.14 as an off-white solid which
was taken to deprotection. HRMS (ESI) m/z calcd for C20H23ClN6O5S2
526.0859 found 527.0939 [M+H]+.
52
PMB protected amine 2.14 was taken in round bottomed flask and
cooled to 0 oC in an ice bath followed by the addition of neat TFA (5
mL). The resulting solution was stirred at room temperature for 2
hours. Upon completion of the reaction the solvent was removed under
reduced pressure and the residue obtained was dissolved in DCM and
subjected to rotary evaporation to azeotropically remove the residual
trifluoroacetic acid (3×15 mL). The crude product obtained was
precipitated using diethyl ether to get pure compound 2.15 as an off-
white solid. (0.057g, 36.5%) 1H NMR (DPX 250 MHz, DMSO-d6) 8.6 (s,
2H), 8.3 (s, 1H), 8.1 (s, 2H), 4.1 (s, 3H), 2.9-2.6 (m, 4H), 2.1 (s, 3H),
HRMS (ESI) m/z calcd for 406.0285 C12H15ClN6O4S2 found 407.0489
[M+H]+.
2-N(chloromethanesulfonyl)-aminopyrimidine (2.16): To a
stirred solution of 2-aminopyrimidine (2.00 g, 21.0 mmol) in pyridine
(25 mL) was added chloromethanesulfonyl chloride (3.29 g, 22.08
mmol) over a period of ten minutes. Progress of the reaction was
monitored by TLC (10% MeOH, 90% EtOAc). Upon completion of the
53
reaction, solvent was evaporated under reduced pressure and the
residue was taken in methanol and evaporated to azeotropically
remove traces of pyridine. The same was repeated three times with 20
mL methanol each time. The crude product was subjected to flash
column chromatography on silica gel using 1:9 MeOH/EtOAc as eluent
to get a pure off-white solid 2.16 (2.73 g, 62.8 %). 1H NMR (Inova
400 MHz, CDCl3) δ 12.11 (s, 1H), 8.61 (d, J=12.63), 7.14 (t, 1H,
J=12.63), 5.28 (s, 2H); 13C NMR (Inova 400 MHz, CDCl3) δ 159.25,
157.39, 116.16, 56.06; HRMS (ESI) m/z calcd for C5H6ClN3O2S
206.9869, found 207.9952, [M+H]+ 229.9770 [M+Na]+.
N-(5-iodo-pyrimidin-2-yl)-chloromethanesulfonamide (2.17):
Same procedure as followed for the synthesis of 2.2 to get pure iodide
2.17 as an off white solid in good yields (86%). 1H NMR (DPX 250 MHz,
CDCl3) δ 8.77 (s, 2H), 7.36 (s, 1H), 5.44 (s, 2H).
54
N-Benzyl-N-(5-iodo-pyrimidin-2-yl)-chloromethanesulfonamide
(2.18):- To a stirred solution of above iodide 2.17 (2.0 g, 6.69 mmol),
KI (0.11 g, 0.67 mmol) and potassium carbonate (1.85 g, 13.38
mmol) in 50 mL of dry DMF under nitrogen atmosphere was added
benzyl bromide at room temperature. Upon completion of the reaction,
solvent was evaporated under vacuo and the residue was dissolved in
ethyl acetate (80 mL) and the same is washed with water (2×15 mL)
and brine (2×15 mL), successively. Organic layer was dried over
anhydrous sodium sulfate and the solvent was evaporated under
vacuo. The crude was subjected to the flash chromatography on silica
gel using EtOAc/Hexane (2:8) as eluent to give pure compound 2.18
as a colorless liquid (81% yield). 1H NMR (DPX 250 MHz, CDCl3) δ 8.71
(s, 2H), 7.3 (m, 5H), 5.3 (s, 2H), 3.34 (s, 3H); 13C NMR (DPX 250
MHz, CDCl3) δ 163.02, 162.99, 137.05, 127.86, 127.66, 84.13, 49.39,
42.92.
55
L-Serine methyl ester hydrochloride (2.19): To a stirred slurry of
L-serine (15.0 g, 143 mmol) in 100 mL of methanol was added SOCl2
(12 mL) at 0 oC over a period of 1 hr. The resulting clear solution was
left to come to room temperature and continued stirring for another 20
hrs before concentrating under reduced pressure. Excess HCl was
azeotropically removed using methanol (3×60 mL) The compound
obtained was dried under high vacuum overnight to give pure product
L-Serine methyl ester hydrochloride 2.19 as white solid (22.0 g, ~
quantitative yields) 1H NMR (DPX 250 MHz, DMSO-d6) δ 8.60 (s, 3H),
5.63 (s, 1H), 4.07 (t, 1H), 3.82 (d, 2H), 3.72 (s, 3H); 13C NMR (DPX
250 MHz, DMSO-d6) δ 168.43, 59.37, 54.32, 52.71.
N-(tert-butoxycarbonyl)-L-serine methyl ester (2.20):- To an ice
cold solution of ester 2.19 (22g, 142 mmol) in THF:H2O (3:1, 100 mL)
56
was added potassium carbonate (20.8g, 150 mmol) followed by Di tert
butyl dicarbonate (35.9 g, 164.5 mmol). The resulting reaction was
stirred vigorously for 23 hrs at room temperature. Upon completion
the reaction mixture was concentrated under reduced pressure and the
obtained crude was extracted into ethyl acetate (3×75 mL) from
saturated brine solution. Organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The obtained crude
was flash chromatographed on short column to get pure product N-
(tert-butoxycarbonyl)-L-serine methyl ester 2.20 as colorless viscous
oil. (30.76 g, 98.8% yield). 1H NMR (DPX 250 MHz, CDCl3) δ 5.72 (d,
1H), 4.49 (s, 1H), 4.04 (d, 2H), 3.91 (s, 3H), 3.19 (s, 1H), 1.58 (s,
9H); 13C NMR (DPX 250 MHz, CDCl3) δ 171.49, 155.80, 80.32, 55.69,
52.65, 28.28; LCMS (ESI) m/z calcd for C9H17NO5 219.11, found 242.1
[M+Na]+.
N-(tert butoxycarbonyl)-O-(p-toluenesulfonyl)-L-serine methyl
ester (2.21):- To an ice cold solution of N-(tert-butoxycarbonyl)-L-
serine methyl ester 2.20 (1.4g, 6.38 mmol) in dry DCM (50 mL) were
57
added 4-dimethylaminopyridine (0.073g, 0.6 mmol) 4-toluene sulfonyl
chloride (1.22g, 6.38 mmol) and triethylamine (0.64 g, 6.38 mmol)
successively The reaction was monitored for the progress by thin layer
chromatography. After completion of reaction the reaction mixture was
concentrated under reduced pressure and the crude was dissolved in
DCM and washed with brine solution. Organic layers were mixed and
dried over Na2SO4 before flash chromatography to get pure compound,
N-(tert butoxycarbonyl)-O-(p-toluenesulfonyl)-L-serine methyl ester
2.21 1.66g (69.6 %). 1H NMR (DPX 250 MHz, CDCl3) δ 7.7 (d, 2H,
J=8.0), 7.3 (d, 2H, J=8.0), 5.2 (d, 1H, J=8.0), 4.5-4.1 (m, 2H), 3.62
(s, 3H), 2.38 (s, 3H), 1.35 (s, 9H); 13C NMR (DPX 250 MHz, CDCl3) δ
168.98, 145.16, 132.37, 129.96, 128.03, 52.97, 28.12, 21.44.
N-(tert-Butoxycarbonyl)-β-iodoalanine methyl ester (2.22): To
a solution of N-(tert butoxycarbonyl)-O-(p-toluenesulfonyl)-L-serine
methyl ester 2.21 (0.87 g, 2.34 mmol) in acetone (25 mL) was
charged NaI (0.53g, 3.52 mmol) under stirring at room temperature.
58
The resulting solution was stirred at same temperature for 10 hrs
before concentrating under reduced pressure. The crude product
obtained was subjected to flash chromatography on silica gel column
using EtOAc: Hexane (1:9) as eluent to get pure product N-(tert-
butoxycarbonyl)-β-iodoalanine methyl ester 2.22 as colorless viscous
liquid (0.58g, 75.0%)
2.6 References
1. Drews, J. Drug discovery: A Historical Perspective. Science.
2000, 287, 1960-1964..
2. Louis Weinstein, Te-Wen Chang, James B. Hudson, Walter Hartl.
The concurrent use of sulfonamides and antibiotics in the
treatment of infections: In vivo and in vitro studies of the effect
of sulfonamide-antibiotic combinations of the emergence of drug
resistance, Ann. N. Y. Acad.Sci.1958, 69, 3, 408-416.
3. Gerhard Domagk. Further Progress in Chemotherapy of bacterial
infections. Nobel Lecture, 1947, December 12.
4. Maren, T. H., and Conroy, C. W. A new class of carbonic
anhydrase inhibitor, 1993, 268, 2623-2639.
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5. Scozzafava, A. Owa, T., Mastrolorenzo, A, and Supuran, C. T.
Anticancer and antiviral sulfonamides. Curr. Med. Chem 2003,
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6. Owa, T., Yoshino, H., Okauchi, T., Yoshimatsu, K., Ozawa, Y.,
Sugi, N. H., Nagasu, T., Koyanagi, N., and Kitoh, K Discovery of
novel antitumor sulfonamides targeting G1 phase of the cell
cycle, J. Med. Chem. 1999, 42, 3789-3799.
7. Yoshino, H.; Ueda, N.; Niijima, J.; Sugumi, H.; Kotake, Y.;
Koyanagi, N.; Yoshimatsu, K.; Asada, M.; Watanabe, T.; Nagasu,
T.; Tsukahara, K.; Iijima, A.; Kitoh, K. Novel sulfonamides as
potential, systemically active antitumor agents. J. Med. Chem.
1992, 35, 2496-2497.
8. (a) Kalgutkar, A. S. Selective Cyclooxygenase-2 Inhibitors as
Nonulcerogenic Antiinflammatory Agents. Exp. Opin. Ther.
Patents 1999, 9, 831-849. (b) Reitz, D. B.; Isakson, P. C.
Cyclooxygenase-2 Inhibitors. Current Pharm. Design 1995, 1,
211-220. (c) Carter, J. S. Recently Reported Inhibitors of
Cyclooxygenase-2. Exp. Opin. Ther. Patents 1997, 8, 21-29.
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9. Ghosh, K. A., Swanson, M. L., Cho, H., Leschenko, S., Hussain,
A. K., Kays, S., Walker, E. D., Koh, Y., and Mitsuya, H.
Structure-based design: Synthesis and biological evaluation of a
series of novel cyclo-amide derived HIV-1 protease inhibitors. J.
Med. Chem. 2005, 48, 3576-3585.
10. Koyanagi, N., Nagasu, T., Fujita, F., Watanabe, T., Tsukahara,
K., Funahashi, Y., Fujita, M., Taguchi, T., Yoshimo, H., and
Kitoh, K. In vivo tumor growth inhibition produced by a novel
sulfonamide, E7010, against rodent and human tumors, Cancer
Res. 1994, 54, 1702-1706.
11. Hung, D. T.; Jamison, T. F., Schreiber, S. L. Understanding and
controlling the cell cycle with natural products Chem. Biol 1996,
3, 623-639.
12. Robert, C. Shepherd and Catherine, E. Fellows. The Iodination of
Certain Phenylsulfonamido- and Amino-heterocylces, J. Am.
Chem. Soc. 1948, 70(1) 157-160.
13 S.Etienne et al, Preparation and characterization of a quinine-
funtioanalised polythiophene film on a modified electrode.
61
14 M. Yamato, Y. Takeuchi, and Y. Ikeda, Heterocycles, 1987, 26,
191.
15 Shaik, N. S.; Gajare, a. S.; Deshpande, V. H.; Bedekar, A. V.;
Tetrahedron Lett 2000, 41, 385.
16 (a) Green, T. W.; Wuts, P. G. M. In Protective Groups in Organic
Syntheis, 3rd ed; Wiley: New York, 1999. (b) Carpino, L.A, Acc.
Chem. Res, 1973, 6, 191.
17 Manjinder, S. Lall.; Yeeman, K. Ramtohul.; Michael, N. James,
and John, C. Vederas. Serine and Threonine β-Lactones: A New
Class of Hepatitis A Virus 3C Cysteine Protease Inhibitors. J. Org.
Chem. 2002, 67, 1536-1547.
18 Park, J.; Fu, H.; Pei, D. Peptidic Aldehydes as Reversible
Covalent Inhibitors of Src Homology 2 Domains. Biochemistry.
2003, 42 (17), 5159-5167.
62
19 Kates, S. A.; Albericio, F. Solid-Phase Synthesis: A Practical
Guide. Marcel Decker. Inc Ed 2000, ISBN 0-8247-0359-6.
20 Prof. Jerry Wu at Moffitt Cancer Center collaborated with us for
testing compounds
21 A. Acero-Alarcón, T. Armero-Alarte, J.M. Jordá-Gregori, C. Rojas-
Argudo, E. Zaballos-García, J. Server-Carrio, F.Z. Ahjyaje and J.
Sepúlveda-Arques, Synthesis. 1999, 12, 2124-2130.
22 Shepherd, R. G.; Fellows, C. E. The Iodination of Certain
Phenylsulfonamido- and Amino-heterocylces. J. Am. Chem. Soc.
1948, 70, 157-160.
23 Bergman, J.; Ola Norrby, P.; Sand, P. Tetrahedron, 1990, 46,
6113.
24 Xing-Guo Zhang, Mu-Wang Chen, Ping Zhong, Mao-Lin Hu.
Regio- and stereo-specific preparation of (E)-1-aryl-3,3,3-
trifluoro-1-iodo-propenes and their palladium-catalyzed reaction
with terminal alkynes. Journal of Fluorine Chemistry 2008, 129,
335-342.
63
25 Stéphanie Étienne, Muriel Matt, Thierry Oster, Mohammad
Samadi, Marc Beley., Preparation and characterization of a
quinone-functionalised polythiophene film on a modified
electrode. Application to the potentiometric determination of
glutathione and cysteine concentrations. Tetrahedron, 2008 64
(40) 9619-9624.
26 Teschner, D. et al. The Roles of Subsurface Carbon and
Hydrogen in Palladium-Catalyzed Alkyne Hydrogenation.
Science, 2008, 320, 86-89.
27 Williams, A. L.; Dandepally, S. R.; Kotturi, S. V. A p-
methoxybenzyl (PMB) protection/deprotection approach toward
the synthesis of 5-phenoxy-4-chloro-N-(aryl/alkyl) thiophene-2-
sulfonamides. Mol Divers. 2009
28 Wuts, P. G. M.; Greene, T. W. Protective Groups in Organic
Synthesis. 4th Ed, 2007, John Wiley & Sons, Inc. New Jersey
29 Shaikh, N. S.; Gajare, A. S.; Deshpande, V. H.; Bedekar, A. V. A
mild procedure for the clay catalyzed selective removal of the
64
tert-butoxycarbonyl protecting group from aromatic amines. Tet.
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30 Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchel, A. R.
VOGEL’s Textbook of Practical Organic Chemistry. 4th Ed. 1989,
ISBN 81-297-0263-0, Pearson Education, India.
31 Yoshida, Y.; Sakakura, Y.; Aso, N.; Okada, S.; Tanabe, Y.
Practical and efficient methods for sulfonylation of alcohols using
Ts(Ms)Cl/Et3N and catalytic Me3H.HCl as combined base:
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32 Richard F.W. Jackson and Manuel Perez-Gonzalez. Synthesis of
N-(tert-butoxycarbonyl)-β-iodoalanine methyl ester: A useful
building block in the synthesis of nonnatural α-amino acids via
palladium catalyzed cross coupling reactions. Organic Syntheses,
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butoxycarbonyl)-β-iodolanine methyl ester: a useful building
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65
66
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77-88.
CHAPTER THREE
SYNTHESIS OF α–ARYL α, β-EPOXY CARBOXYLATES,
PHOSPHONATES AND THEIR BIOLOGICAL ACTIVITY AGAINST
TYROSINE PHOSPHATASES
3.1. General introduction
Carboxylates are a very important class of compounds to both
biologists and chemists as they play some very significant roles in
biology. One important class of compounds with carboxylic acid
functional group is amino acids which are indispensable to the living
organism whether they are primitive organisms or advanced
organisms.1,2 2-Hydroxypropanoic acid also known as lactic acid plays
a role in several biochemical processes and was first isolated in 1780
by Swedish chemist Carl Wilhelm Scheele. In animals, it is produced
from another carboxylic acid peruvate constantly.3 (Figure 3.1).
Similarly, phosphates are also very significant as they play very crucial
roles in the biology as in the formation of adenosine tri-phosphate
from adenosine di-phosphate and the phosphorylation of most of the
enzymes to get into the right 3D conformation so as to perform the
assigned function in the body.4
66
Figure 3.1 Conversion of pyruvate to lactate in respiratory cycle
Our current interest is to synthesize phosphonates and
carboxylates as phosphate mimics of both epoxy and aziridine
derivatives and test their activities to develop the inhibitors for
tyrosine phosphatases mainly concentrating on SHP-2 and PTP1B
which has been briefly explained in chapter one. Some of the
important carboxylates which were tested for activity against PTP1B
were shown in Figure 3.2. Ertiprotafib of Wyeth-Ayerst reached clinical
trials and failed to go further because of efficacy issues.5
67
Figure 3.2 Some of the phosphatase inhibitors with carboxy functionality6
3.2. Phosphonates as tyrosine phosphatase inhibitors
Synthesis of phosphonate ester 3.4 was discussed and the
activity is explained in the following sections of this chapter.
3.3. Carboxylates as tyrosine phosphatase inhibitors
68
Synthesis of carboxylates was discussed and the activity is
explained in the following sections of this chapter.
3.4. Results and discussion
3.4.1 Synthesis of α-aryl α,β-epoxy phosphonates
Synthesis of the phosphonates started from commercially
available benzoyl chloride which was reacted with triethyl phosphite to
form α-keto phosphonate ester 3.2. Phosphonate 3.2 was epoxidized
with trimethyl sulphonium iodide prepared in our lab to get α,β-epoxy
phosphonate ester 3.3 in good yields. Epoxy phosphonate was
subjected to mono deprotection with LiBr in 4-methyl-2-pentanone at
80 oC to get compound 3.4 in good yields.7 (Scheme 3.1)
Scheme 3.1 Synthesis of α,β-epoxy phosphonate 3.4
69
The direct di-deprotection of 3.3 failed to give the desired
phosphonic acid by using the same conditions at higher temperatures.
The mono deprotected compound starts to precipitate as lithium salt
3.4 and so does not give the product. We then converted the di alkyl
phosphonate to di silyl phosphonate 3.5 using trimethyl silyl chloride
(TMSCl) in dichloromethane as solvent which can be easily hydrolyzed
to the corresponding epoxy phosphonic acid 3.6 or tested without
deprotection as it can get deprotected at physiological conditions.8
Scheme 3.2 Synthesis of α,β-epoxy phosphonic acid 3.6
3.4.2. Synthesis of α-aryl-α,β-epoxy carboxylates
Synthesis of carboxylates started from the commercially
available aromatic aldehydes and alpha phenyl methyl acetate. Alpha
phenyl methyl acetate was converted to diaza ester 3.7 employing
diazo transfer reaction with p-ABSA as diaza transfer reagent and DBU
as base in excellent yields.9 Diazo-ester 3.7 was reacted with various
in house aldehydes to get α,β-epoxy carboxylates in very good yields.
The product with benzaldehyde was trans as reported in literature.10
70
We selected aromatic aldehydes with various electron donating,
electron withdrawing and alkyl, halo and alkoxy groups so that we can
study the effects of substituents on the activity against selected
phosphatases.
Scheme 3.3 Synthesis of α,β-epoxy α-aryl carboxylates 3.9a-g
The methyl ester was deprotected using standard conditions with
LiOH as base in THF: H2O mixture to get carboxylic acid 3.9 in
excellent yields.
71
Compound 3.9a was also prepared by using n-BuLi/TMEDA/CO2
reagents from (trans)-1,2-Di-phenyl oxirane to see if it gives
exclusively trans product (Boxed in Scheme 3.5). The same conditions
with formaldehyde and its equivalents did not give the desired product
3.8h as shown in Scheme 3.4 instead gave the dimerized derivative of
diazo-ester 3.8i which was confirmed using NMR techniques as well as
crystal structure studies.11 The directed lithiation with BuLi/TMEDA and
subsequent reaction with CO2 as electrophile were not reported
anywhere in the literature to the best of our knowledge. Whereas
different electrophiles like alkyl iodides give the corresponding
derivatives in excellent yields and were reported in literature.12
Scheme 3.4 Failed attempts to synthesis epoxy carboxylates with methanal equivalents
72
3.4.3 Synthesis of α,β-aziridino carboxylates
The results of directed lithiation on epoxide 3.10 and trapping
with carbon dioxide prompted us to apply the same methodology to
make aziridine derivatives. For this approach, trans-stilbene was
epoxidized using m-CPBA as epoxidizing agent in chloroform at 0 oC to
get trans-epoxide 3.10 in very good yields. The epoxide was cleaved
to get trans-azido alcohol 3.11 using sodium azide and ammonium
chloride as reported in the literature. The azido alcohol was refluxed in
acetonitrile in the presence of triphenylphosphine to obtain aziridine
3.12 in excellent yields.13 The attempted directed lithiation with n-
BuLi/TMEDA followed by reaction with carbon dioxide did not give any
desired product. Changing n-BuLi with s-BuLi and t-BuLi did not
improve the results. Thus, we changed the strategy and the free NH
group was protected as the Boc derivative using di-tert-butoxy
anhydride in the presence of DMAP in dichloromethane as solvent gave
excellent yields of aziridine 3.13.14 The product was then subjected to
the same directed lithiation conditions but we were only able to run
mass spectral analysis as the yields were dismal. (Scheme 3.5)
73
Scheme 3.5 Synthesis of α,β-aziridino carboxylate 3.14
This led us to apply the same Rhodium catalyzed reaction
conditions applied above in making carboxylates 3.8a-g. For this, we
needed imines with suitable groups on amine so that it is easy to
remove after the insertion reaction, which will be explained in this
report shortly.
Benzaldehyde was reacted with 40% methylamine in water at 0
oC to get N-methyl imine 3.16 in excellent yields.15 The reaction with
4-methoxybenzylamine in presence of anhydrous sodium sulfate in
dichloromethane at room temperature gave very good yields of imine
74
3.17. The rhodium catalyzed insertion reaction with diazo-ester 3.17
did not give the desired product and we could not find any reason
behind this. To find out if the free NH group is having any effect on the
reaction conditions we attempted the same reaction conditions on
styrene and the reaction worked perfectly to give 3.21 in very good
yields as reported in the literature. So the protecting group has been
changed to benzyl and for that benzaldehyde was protected as imine
3.18 and was subjected to obtain N-benzyl aziridine carboxylate 3.19
in decent yields. The ester was subjected to base hydrolysis using
excess lithium hydroxide in tetrahydrofuran and water mixture as
solvent to get the corresponding carboxylic acid 3.20 in moderate
isolated yields. (Scheme 3.7)16
Scheme 3.6 Synthesis of N-alkyl α,β-aziridino carboxylate 3.19
75
Scheme 3.7 Synthesis of N-benzyl aziridine carboxylate 3.20
Scheme 3.8 Synthesis of α,β-diaryl α-cyclopropane carboxylate
3.5. Biological activity studies
IC50 was defined as the concentration of compound that caused a
decrease of 50% in magnitude in the PTP activity. 2-Aminopyrimidine
chlorides, sulfonamides and α-aryl, α,β-epoxy carboxylates and
phosphates synthesized were tested for activity of inhibition for IC50
using a human recombinant GST-Shp-2 PTP domain protein. 6,8-
Difluoro-4-methylumbelliferyl phosphate (DiFMUP) was used as the
76
substrate. Testing was performed in duplicates at room temperature in
black, half area 96-well plates. Incubation was carried out at room
temperature for 20 min in a 75 µL reaction mixture containing 25 mM
Hepes, pH 7.0, 50 mM NaCl, 1 mM DTT, 0.01% Triton X-100, 40 µM
DiFMUP, 3% DMSO or compound. Fluorescent signal was measured at
excitation and emission wavelengths of 355 nm and 460 nm
respectively. For IC50 determination, eight concentrations of each
compound at one third dilutions were tested. The ranges of compound
concentrations used in PTP assay were determined from preliminary
trials. According to the results obtained epoxy carboxylates exhibited
good inhibitory activity among the compounds synthesized.
Preliminary results show that these compounds work as noncovalent
inhibitors.
Compounds 3.9d and 3.9c showed activity against Shp-2 protein
phosphatase with IC50 values 5.6 mM and 20.8 mM respectively. Epoxy
carboxylate 3.9a without any aromatic substitutions showed good
activity against the same target with IC50 value of 0.0069 mM (6.9
µM).
3.6. Conclusion and future directions
Synthesis of some epoxy and aziridino carboxylates and
phosphonates was performed and some of them were tested against
PTPs and we were excited by some of relatively promising inhibitors
77
against SHP-2 phosphatase. The obtained compounds can be modified
by using the same methodology mentioned in chapter-2 of this writing
to make amino acid derivatives of the same carboxylates and put into
small peptides which can increase the selectivity many times.
Compound 3.9a shows good activity against Shp-2 phosphatase and
this kind of epoxy esters are more stable than the simple epoxides and
it is interesting to see if the activity can be increased simultaneously
by substituting with pyrimidine units and making the second
generation compounds. It can be put into small peptides and can be
more selective at the same time more active against Shp-2
phosphatase.
3.7 Experimental Procedures
Diethyl-(phenylcarbonyl)-phosphonate (3.2)
To an ice cold solution of benzoyl chloride (4.0 g, 28.4 mmol) in 25 mL
of dichloromethane was added triethyl phosphite (4.7 g, 28.4 mmol)
dropwise. Reaction was left to come to room temperature and stirred
for a further 12 hours. After the complete consumption of starting
material, 50 mL of dichloromethane was added, and the organic layer
78
was washed with saturated sodium bicarbonate solution (3×15 mL)
followed by saturated brine solution (3×15 mL). The extract was dried
over anhydrous MgSO4 and evaporated in vacuo. The residue was
purified by column chromatography on silica gel using EtOAc/Hexanes
(1:9) as eluent to give the pure product 3.2 as a greenish yellow
liquid. (4.9 g, 72 %). 1H NMR (DPX 250 MHz, CDCl3) δ 8.3 (m, 2H), 7.6
(t, 1H), 7.5 (m, 2H), 4.3 (q, 4H), 1.4 (t, 6H); 13C NMR (DPX 250 MHz,
CDCl3) δ 200.4, 136.0, 134.7, 129.8, 128.8, 63.9, 16.3; HRMS (ESI)
m/z calcd for C11H15O4P is 242.070 found 243.078 [M+H]+.
Phosphonic acid, (2-phenyloxiranyl)-, diethyl ester (3.3)
A solution of triphenylphosphine methyl bromide (1.55 g, 4.33 mmol)
and DIEA (57 µL, 0.41 mmol) in THF (15 mL) was cooled to -76 oC
before adding n-BuLi dropwise. After briefly stirring at -76 oC for about
15 minutes, the reaction was brought to room temperature and further
stirred for an hour before cooling back to -76 oC at which temperature,
the phosphate ester (1.0 g, 4.13 mmol) was added. The reaction was
left to come to room temperature and further stirred for 15 hours
79
before quenching with 1N HCl solution at ice cold temperature. The
product was extracted into diethyl ether (3×25 mL). All organic layers
were combined and washed with brine solution (3×15 mL) and dried
over anhydrous Na2SO4 and evaporated under reduced pressure to get
crude product which on subjecting to flash column chromatography
gave pure product 3.3 as colorless liquid (0.66 g, 67 %).1H NMR (DPX
250 MHz, CDCl3) δ 7.9-7.3 (m, 5H), 4.0 (q, 4H), 1.25 (t, 6H); 13C NMR
(DPX 250 MHz, CDCl3) δ 152.25, 134.37, 129.10, 128.40, 125.21,
97.32, 64.59, 16.07; HRMS (ESI) m/z calcd for C12H17O4P is 256.0864,
found 257.0 [M+H]+
(2-Phenyl-oxiranyl)-lithium phosphonate mono-ethyl ester
(3.4)
To a solution of diethyl phosphonate 3.3 (0.1 g, 0.39 mmol) in 4-
methyl-2-pentanone (5 mL), lithium bromide (0.034 g, 0.39 mmol),
was added and the mixture was stirred at 70 oC. After a few minutes
lithium bromide dissolved and a white precipitate started to
precipitate. Heating was continued for about 2 hours. The solvent was
80
removed under vacuo and ether (20 mL) was added to the residue.
The product was collected by filtration and washed with another 10 mL
of ether. No further purification was needed as the compound was
pure on TLC and NMR studies. The pure compound 3.4 was obtained
as white solid. (0.05 g, 48%).1H NMR (DPX 250 MHz, CDCl3) δ 7.39-
7.24 (m, 5H), 5.0 (d, 2H), 3.7 (q, 2H, J=7.50), 2.5 (s, 1H), 1.2 (t, 3H,
J=7.5); 13C NMR (DPX 250 MHz, CDCl3) δ 153.13, 137.10, 127.95,
127.82, 124.83, 92.74, 59.99, 16.57 ; HRMS (ESI) m/z calcd for
C10H13O4P is 228.055, found 227.01 [M-H]-.
Diazo-phenyl-acetic acid methyl ester (3.7):
To a stirred solution of 4-acetamidobenzenesulfonyl azide (p-
ABSA, 24.8 g, 20.0 mmol) in 45 mL of dry acetonitrile was charged
with methyl phenyl acetate (2.5 g, 17 mmol) followed by the dropwise
addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 3.55 g, 23.3
mmol) at ambient temperatures. Reaction mixture gradually turned
into reddish from yellow. After the completion of the reaction by TLC,
in about 8 hours, the reaction was diluted with 20 mL of water.
Product was extracted with ether (3×15 mL). Organic fractions were
81
combined and washed with 10% sodium bicarbonate solution (3×10
mL), followed by saturated brine solution (3×10 mL). Organic layer
was dried over anhydrous sodium sulfate before evaporating under
reduced pressure to give crude product. Flash chromatography on
silica gel using EtOAc/Hexane (1:9) as eluent afforded pure diaza ester
3.7 as a red liquid (2.69 g, 93%). 1H NMR (DPX 250 MHz, CDCl3) δ
7.4-7.3 (m, 2H), 7.3-7.2 (m, 2H), 7.1-7.0 (1H), 3.75 (s, 3H); 13C NMR
(DPX 250 MHz, CDCl3) δ 165.58, 129.29, 128.6, 127.13, 125.50,
123.95, 52.04.
General Procedure for Rh2(OAc)4 catalyzed carbene insertion
reaction:
To a flame dried 100 mL 3 necked round bottom flask, Rh2(OAc)4 was
charged under nitrogen flow. To the same freshly distilled
dichloromethane was charged followed by the addition of aldehyde.
The resulting solution was brought to 45 oC and a solution of diazo
ester 3.7 in dry dichloromethane was added over a period of 1.5
82
hours. The reaction was monitored for the progress by thin layer
chromatography. After the disappearance of the aldehyde the reaction
was brought to ambient temperatures and the reaction mixture was
filtered through Celite and the same was concentrated under reduced
pressure. The crude product obtained was subjected to flash column
chromatography on silica gel to get corresponding epoxy carboxylates
(racemic) 3.8a-g in pure form. (TLC solvent 30% EtOAc in 70%
Hexane)
2,3-diphenyl-oxirane-2-carboxylic acid methyl ester (3.8a)
colorless solid, 88% yield 1H NMR (DPX 250 MHz, CDCl3) δ 7.6 (m,
2H), 7.4-7.3 (m, 8H), 4.1 (s, 1H), 3.5 (s, 3H); 13C (DPX 250 MHz,
CDCl3) δ 167.2, 134.7, 133.7, 128.9, 128.7, 128.6, 128.4, 126.2,
126.0, 67.1, 65.9, 52.3.
83
3-(4-chloro-phenyl)-2-phenyl-oxirane-2-carboxylic acid methyl
ester (3.8b) white solid, 67% yield. 1H NMR (DPX 250 MHz, CDCl3) δ
7.6-7.5 (m, 2H), 7.35-7.30 (m, 3H), 7.25-7.2 (m, 4H), 4.04 (s, 1H),
3.49 (s, 3H)
3-(4-bromo-phenyl)-2-phenyl-oxirane-2-carboxylic acid methyl
ester (3.8c) white solid, 91% yield. 1H NMR (DPX 250 MHz, CDCl3) δ
7.9 (d, 2H), 7.7 (d, 2H), 7.4-7.2 (m, 5H), 4.2 (s, 1H), 3.7 (s, 3H), 3.6
(s, 3H); LCMS (ESI) m/z calcd for C16H13BrO3 is 332.0, found 332.9 &
334.9 [M+2+H]+
3-(4-methoxy-phenyl)-2-phenyl-oxirane-2-carboxylic acid
methyl ester (3.8d) white solid, 86% yield. 1H NMR (DPX 250 MHz,
CDCl3) δ 7.63 (d, 2H), 7.40-7.25 (m, 5H), 6.95-6.85 (d, 2H), 4.08 (s,
84
1H), 3.78 (s, 3H), 3.56 (s, 3H); HRMS (ESI) m/z calcd for C17H16O4 is
284.10, found 285.1 [M+H]+.
3-(4-nitro-phenyl)-2-phenyl-oxirane-2-carboxylic acid methyl
ester (3.8e) yellowish solid, 63% yield. 1H NMR (DPX 250 MHz,
CDCl3) δ 8.4 (d, 2H), 8.1 (d, 2H), 7.8-7.1 (m, 5H), 4.1 (s, 1H), 3.4 (s,
3H); 13C (DPX 250 MHz, CDCl3) δ 165.3, 134.2, 130.6, 129.2, 128.5,
127.1, 126.9, 124.7, 124.5, 66.4, 63.3, 52.4,; HRMS (ESI) m/z calcd
for C16H13NO5 is 300.08, found 317.11 [M+NH4]+
3-(2-methoxy-phenyl)-2-phenyl-oxirane-2-carboxylic acid
methyl ester (3.8f) colorless solid, 87% yield. 1H NMR (DPX 250
MHz, CDCl3) δ 7.6 (m, 2H), 7.45-7.25 (m, 4H), 6.95-6.75 (m, 3H),
4.32 (s, 1H), 3.73 (s, 3H), 3.40 (s, 3H); HRMS (ESI) m/z calcd for
C16H17O4 is 284.10, found 285.10 [M+H]+
85
3-(2-nitro-phenyl)-2-phenyl-oxirane-2-carboxylic acid methyl
ester (3.8g) yellowish solid, 72% yield. 1H NMR (DPX 250 MHz,
CDCl3) δ 8.2-8.1 (d, 1H), 7.8-7.6 (m, 4H), 7.4-7.2 (m, 4H), 4.6 (s,
1H), 3.3 (s, 3H); HRMS (ESI) m/z calcd for C16H13NO5 is 299.07, found
300.08 [M+H]+, 317.11 [M+NH4]+
General procedure for the deprotection of methyl ester to
carboxylic acid:-
To a solution of α,β-epoxy methyl carboxylate 3.8a-g in
tetrahydrofuran-water mixture (~1:1) was added excess LiOH solution
86
(1N) at 0 oC under stirring. Progress of the deprotection was
monitored by thin layer chromatography. Upon completion of the
deprotection the reaction mixture was subjected to rotary evaporation
to remove organic solvent. The resulting basic solution was washed
with ethyl acetate to remove any unreacted ester and the aqueous
layer was cooled in an ice bath before neutralizing to pH ~7.0. The
same was extracted into ethyl acetate (3×40 mL) and the organic
portions were combined together and dried over anhydrous Na2SO4
before concentrating under reduced pressure. The product was pure
enough most of the times but needed to do flash column
chromatography to get the corresponding carboxylic acid 3.9a-g
(racemic). (TLC solvent 10% MeOH in 90% EtOAc)
2,3-diphenyl-oxirane-2-carboxylic acid (3.9a) white solid, 76%
yield. 1H NMR (DPX 250 MHz, CDCl3) δ 11.9 (s, 1H), 7.6 (m, 2H), 7.4-
7.3 (m, 3H), 4.0 (s, 1H); HRMS (ESI) m/z calcd for C15H12O3 is
240.08, found 241.09 [M+H]+.
87
3-(4-chloro-phenyl)-2-phenyl-oxirane-2-carboxylic acid (3.9b)
off-white solid, 80% yield. 13C NMR (DPX 250 MHz, CDCl3) δ 181.9,
137.8, 134.9, 132.1, 128.6, 127.8, 127.6, 127.3, 126.3; HRMS (ESI)
m/z calcd for C15H11ClO3 is 274.04, found 275.04 [M+H]+.
3-(4-bromo-phenyl)-2-phenyl-oxirane-2-carboxylic acid (3.9c)
white solid 81% yield. 1H NMR (DPX 250 MHz, CDCl3) δ 7.45-7.35 (m,
4H), 7.6-7.5 (m, 3H), 7.3-7.2 (m, 2H), 4.4 (s, 1H), 3.3 (bs, 1H); 13C-
NMR 177.96, 133.69, 131.69, 129.41, 129.17, 128.70, 127.90,
127.44, 126.40, 65.48, 40.98.; HRMS (ESI) m/z calcd for C15H11BrO3
is 317.9, found 318.9 [M+H]+
88
3-(4-methoxy-phenyl)-2-phenyl-oxirane-2-carboxylic acid
(3.9d) white solid, 58% yield. HRMS (ESI) m/z calcd for C16H14O4 is
270.09, found 271.09 [M+H]+.
3-(4-nitro-phenyl)-2-phenyl-oxirane-2-carboxylic acid (3.9e)
yellow solid, 62% yield. HRMS (ESI) m/z calcd for C15H11NO5 is
285.06, found 286.07 [M+H]+.
3-(2-methoxy-phenyl)-2-phenyl-oxirane-2-carboxylic acid
(3.9f) white solid, 66% yield. HRMS (ESI) m/z calcd for C16H14O4 is
270.09, found 271.09 [M+H]+.
89
3-(2-nitro-phenyl)-2-phenyl-oxirane-2-carboxylic acid (3.9g)
brown solid, 74.3% yield, 1H NMR (DPX 250 MHz, DMSO-d6) δ 8.2-8.1
(d, 1H), 7.8-7.6 (m, 4H), 7.4-7.2 (m, 3H), 7.6-7.5 (d, 1H), 4.4 (s,
1H); HRMS (ESI) m/z calcd for C15H11NO5 is 285.06, found 286.07
[M+H]+
2,3-diphenyloxirane (3.10)
To an ice cold solution of m-CPBA (65%, 2.7 g, 10.20 mmol) in
chloroform (30 mL) was added a solution of trans-stilbene (1.0 g, 5.55
mmol) in chloroform (30 mL) over a period of 30 minutes. The
reaction was left to come to ambient temperatures and stirred further
until the starting material disappeared. The reaction mixture was
90
diluted with chloroform (40 mL) and the same was washed with
saturated NaHCO3 (3×30 mL) and saturated brine solution (3×20 mL)
successively and the organic layer was dried over anhydrous Na2SO4
before concentrating to get crude product which on further purification
by flash column chromatography gave pure epoxide 3.10 as thick
colorless oil (0.73 g, 67.59%). 1H NMR (DPX 250 MHz, CDCl3) δ 7.47-
7.37 (m, 10H), 3.92 (s, 2H); 13C NMR (DPX 250 MHz, CDCl3) δ 137.1,
128.6, 128.4, 125.6, 62.9; HRMS (ESI) m/z calcd for C14H12O is
196.08, found 197.09.[M+H]+
β-azido-α-phenyl benzeneethanol (3.11)
Trans-stilbene oxide 3.10 (1g, 5.11 mmol), sodium azide (0.76 g, 11.
73 mmol), and NH4Cl were dissolved in 40 mL of MeOH: H2O (4:1).
The resulting clear solution was refluxed for 2.5 hrs and then allowed
to stir at room temperature for additional 8 hrs. Upon completion of
the reaction by TLC dichloromethane and water (25 mL each) were
added to the reaction mixture. The two layers were separated and the
water layer was extracted with dichloromethane (2×25 mL). The
91
organic layers were combined and washed with saturated brine
solution (2×25 mL) before drying over anhydrous MgSO4. Removal of
solvent and further purification of the obtained crude by flash column
chromatography gave pure azido-alcohol 3.11 as light yellow liquid
(0.91 g, 74 %). 1H NMR (DPX 250 MHz, CDCl3) δ 7.3-7.2 (m, 10H), 4.8
(dd, 1H, J12 = 3.00, J13 = 3.50), 4.7 (d, 1H, J=6.75), 2.3 (d, 1H,
J=3.00); 13C NMR (DPX 250 MHz, CDCl3) δ 139.71, 136.01, 128.74,
128.69, 128.41, 128.33, 128.11, 127.10, 77.00, 71.27.
2,3-Diphenylaziridine (3.12)
Azido-alcohol 3.11 (3.6 g, 15 mmol) and PPh3 (4.01 g, 15.3 mmol)
were dissolved in acetonitrile (40 mL) under nitrogen atmosphere and
heated to reflux for 2.5 hrs. Upon completion of the reaction the
solvent was removed under reduced pressure and the resultant crude
was purified by flash column chromatography to get pure aziridine
3.12 as light yellow liquid (2.72 g, 92.6 %). 1H NMR (DPX 250 MHz,
CDCl3) δ 7.32-7.12 (m, 10H), 3.00 (bs, 2H), 1.28 (bs, 1H, NH); 13C
92
NMR (DPX 250 MHz, CDCl3) δ 139.7, 128.7, 127.5, 125.5, 43.7; HRMS
(ESI) m/z calcd for C14H13N is 195.1048 found 196.1185 [M+H]+
1-Aziridinecarboxylic acid,2,3-diphenyl-, 1,1-dimethylethyl
ester (3.13)
To a solution of aziridine 3.12 (0.50 g, 2.6 mmol) in dichloromethane
(25 mL) at ice-cold temperature were added di-tert-butyl dicarbonate
(0.67 g, 3.1 mmol) and DMAP (0.03 g, 0.25 mmol). Reaction was
monitored for the progress by thin layer chromatography. Starting
material completely disappeared in 40 minutes. Solvent was removed
under reduced pressure and the crude was subjected to flash column
chromatography to get pure compound as the colorless solid (0.69 g,
91.7 %). 1H NMR (DPX 250 MHz, CDCl3) δ 7.53-7.47 (m, 10H), 3.92
(s, 2H), 1.33 (s, 9H); 13C NMR (DPX 250 MHz, CDCl3) δ 159.41,
135.55, 128.52, 128.12, 127.03, 81.47, 47.63, 27.66.
2,3-diphenyl, 1-tert-butoxy carbonyl-2-aziridine carboxylic acid
(3.14)
93
To a stirred solution of 1-tert-butoxy carbonyl-2-aziridine 3.13 in
tetrahydrofuran was added TMEDA at -78 oC. To the above solution n-
BuLi was added and the solution stirred at the same temperature for 2
hrs before adding dry ice. The reaction was brought to room
temperature gradually and monitored for the progress by thin layer
chromatography. No significant product was obtained even though
HRMS and LCMS showed the presence of product. Repeated reactions
with s-BuLi and t-BuLi did not result in any improvements.
N-(phenylmethylene)-methanamine (3.16)
A mixture of benzaldehyde (12.0 g, 113 mmol) and excess of 40%
methane amine in water (16 mL, 192.0 mmol) was stirred at 0 oC for 2
hrs and further stirred at ambient temperatures overnight. After the
94
completion of the reaction the product was extracted into ethyl acetate
(3×50 mL). Organic layers were combined washed with brine solution
(3×40 mL) and dried over anhydrous sodium sulfate. The solvent was
removed under reduced pressure and further vacuum purified to get
pure product 3.16 as liquid (13.45 g, 97%) 1H NMR (250 MHz, CDCl3)
δ 8.25 (s, 1H), 7.75 (m, 2H), 7.45 (m, 3H); 13C NMR (250 MHz, CDCl3)
δ 162.50, 136.28, 130.65, 128.64, 127.01, 40.29.
Benzenemethanamine-4-methoxy-N-(phenylmethylene) (3.17)
To a stirred solution of benzaldehyde (1.39 g, 13.1 mmol) and 4-
methoxy benzylamine (1.8 g, 57 mmol) in dichloromethane (30 mL)
was added anhydrous Na2SO4 (1.6 g, 113 mmol) at ambient
temperatures. Reaction was stirred at the same temperature for about
12 hours before filtering the solids off and concentrating under
reduced pressure. The crude product obtained was subjected to flash
column chromatography to get pure compound 3.17 as a colorless
liquid (2.36 g, 80.0%). 1H NMR (DPX 250 MHz, CDCl3) δ 8.21 (s, 2H),
7.68-7.58 (m, 2H), 7.30-7.20 (m, 3H), 7.17-7.08, (d, 2H), 6.80-6.70
95
(d, 2H); 13C NMR (DPX 250 MHz, CDCl3) δ 192.44, 158.76, 136.30,
131.46, 130.80, 129.81, 129.08, 128.68, 128.35, 64.59, 55.32.
Benzyl-benzylidene-amine (3.18)
Same procedure was followed as mentioned for 3.17 benzaldehyde
(6.0 g, 57 mmol) and benzylamine (6.05 g, 56.5 mmol) in
dichloromethane (100 mL) with anhydrous Na2SO4 (16.06 g, 113.1
mmol). 3.18 is colorless liquid. (10.26 g, 92.93 %). 1H NMR (DPX 250
MHz, CDCl3) δ 8.46 (s, 1H), 7.95-7.75 (m, 2H), 7.55-7.25 (m, 6H),
4.90 (s, 2H); 13C (DPX 250 MHz, CDCl3) δ 162.08, 139.33, 136.18,
130.84, 128.67, 128.56, 128.34, 128.04, 127.06, 65.10; LCMS (ESI)
m/z calcd for C14H13N is 195.1048 found 196.10 [M+H]+.
1-Benzyl-2,3-diphenyl-aziridine-2-carboxylicacid methyl ester
(3.19)
96
To a stirred solution of imine (0.886 g, 4.54 mmol) in dry
dichloromethane (15 mL) was added Rh2(OAc)4 (0.02 g, 0.045 mmol)
under nitrogen atmosphere and the reaction mixture was heated to 40
oC. A solution of diazo ester 3.7 (0.80g, 4.54 mmol) in dry
dichloromethane (10 mL) was added over a period of 40 minutes and
the reaction was monitored for progress. After the completion, the
reaction mixture was passed through Celite bed to remove rhodium
salts. The solvent was removed and the crude was flash
chromatographed to get pure compound as yellow solid. (0.426 g,
30.3 %). 1H NMR (DPX 250 MHz, CDCl3) δ 7.75 (m, 3H), 7.65-7.15
(m, 12H), 4.31 (s, 1H), 3.91 (s, 2H), 3.60 (s, 3H); 13C NMR (DPX 250
MHz, CDCl3) δ 165.78, 139.47, 138.04, 132.20, 131.09, 128.89,
128.77, 128.48, 128.37, 128.18, 128.06, 127.61, 127.20, 79.86,
64.34, 52.29, 51.35.HRMS (ESI) m/z calcd for C23H21NO2 343.1572
found 344.1626 [M+H]+.
1-Benzyl-2,3-diphenyl-aziridine-2-carboxylicacid (3.20)
97
Same procedure was used as mentioned for the hydrolysis of ester
3.8a-g except increasing the THF ratio in the solvent mixture.
3.8. References
1. a). Block, R. J. The isolation and synthesis of naturally occurring
α-amino acids. Chem. Rev. 1946, 38, 501-571.
2. Farina, V.; Reeves, J. T.; Senanayake, C. H.; Song, J. J.
Asymmetric synthesis of Active Pharmaceutical Ingredients.
Chem. Rev. 2006, 106, 2734-2793.
3. Chandra Raj, K.; Ingram, L. O.; Furlow, J. A. Pyryvate
decarboxylase. a key enzyme for the oxidative metabolism of
lactic acid by Acetobacter pasteurianus. Arch. Microbiol. 2001,
176, 443-451.
4. Knowles, J. R. Enzyme catalyzed phosphoryl transfer reactions.
Annu. Rev. Biochem. 1980, 49, 877-919.
5. Wa¨ lchli, S.; Curchod, M.-L.; Pescini Gobert, R.; Arkinstall, S.;
Hooft van Huijsduijnen, R. Identification of tyrosine
phosphatases that dephosphorylate the insulin receptor: a
bruteforce approach based on “substrate-trapping” mutants. J.
Biol. Chem. 2000, 275, 9792-9796.
98
6. a). Xing Jiang, Z.; Yin Zhong, Z. Targeting PTPs with small
molecule inhibitors in cancer treatment. Cancer Metastasis Rev.
2008, 27(2), 263-272. b). Kumar, R.; Shinde, R. N.; Ajay, D.;
Sobhia, E. M. Probing Interaction Requirements in PTP1B
Inhibitors: A Comparative Molecular Dynamics Study. J. Chem.
Inf. Model. 2010, 50, 1147-1158.
7. Krawczyk, H. A convenient route for Monodealkylation of Diethyl
Phosphonates Synthetic. Communications. 1997, 27(18), 3151-
3161.
8. Gutierrez, a. J.; Prisbe, E. J.; Rohloff, J. C. Dealkylation of
Phosphonate Esters with Chlorotrimethylsilane. Nucleosides,
Nucleotides & Nucleic Acids. 2001, 20(4-7), 1299-1302.
9. a). Doyle, M. P., McKervey, M. A., and Ye, T. Modern Catalytic
Methods for Organic Synthesis with Diazo Compounds; Wiley-
Interscience: New York, 1998. b) Zollinger, H. Diazo Chemistry I
& II; VCH: New York, 1994. c). Yiu Yu, W.; Tai Tsoi, W.; Zhou,
Z.; Chan, A. C. S. Palladium catalyzed cross coupling Reaction of
Bromides with Diazoesters for Stereoselective Synthesis of (E)-α,
β-Diaryacrylates. Org. Lett. 2009, 11(2), 469-472.
10. a). Lu, C. D.; Chen, Z. Y.; Liu, H.; Hu, W. H.; Mi, A. Q. Highly
Chemoselective 2,4,5-Triaryl-1,3-Dioxolane Formation from
Intermolecular 1,3-dipolar Addition of Carbonyl Ylide with Aryl
99
Aldehydes. Org. Lett. 2004, 6(18), 3071-3074. b). Davies, H. M.
L.; DeMeese, J. Stereoselective synthesis of epoxides by reaction
of donor/acceptor-substituted carbenoids with α,β-unsaturated
aldehydes. Tet Lett. 2001, 42, 6803-6805.
11. Crystal Structure Details and NMRs are available in the
supporting information of this dissertation.
12. a). Capriati, V.; Florio, S.; Luisi, R.; Musio, B. Directed Ortho
Lithiation of N-Alkylphenylaziridines. Org. Lett. 2005, 7(17),
3749-3752. b). Luisi, R.; Capriati, V.; Degennaro, L.; Florio, S.
Oxazolinyloxiranyllithium-Mediated Synthesis of α-Epoxy, β-
Amino Acids. Org. Lett. 2003, 5(15), 2723-2726.
13. Ittah, Y.; Sasson, Y.; Shahak, I.; Tsaroom, S.; Blum, J. A New
Aziridine synthesis from 2-Azido Alcohols and Tertiary
Phosphines. Preparation of Phenanthrene 9,10-Imine. J. Org.
Chem. 1978, 43(22), 4271-4273.
14. Green, T. W.; Wuts, P. G. M. Protective Groups in Organic
synthesis. Wiley-Interscience, New York, 1999, 4th Ed. 518-525,
736-739.
15. Semenov, B. B. et al. diastereoselected alkylation of ketones and
1,3-diketones with N-[1H-indol-3-yl(phenyl)methyl]-N-
methylamine by the Michael reaction. Chemistry of Heterocyclic
Compounds. 2005, 41(6), 730-738.
100
101
16. Doyle, M. P.; Hu, W.; Timmons, D. J. Epoxides and Aziridines
from Diazoacetates via Ylide Intermediates. Org. Lett. 2001,
3(6), 933-935.
CHAPTER FOUR
TOWARDS THE SYNTHESIS OF CYSTEINE BASED PEPTIDE
NUCLEIC ACIDS (CPNAs) AND HYBRIDS
4.1 General introduction
The genetic information of most organisms is encoded in the
sequence of double stranded DNA (dsDNA), also known as the
molecule of life, which is transcribed into mRNA during transcription.
The blocking of transcription has become an attractive target for
therapeutic discovery.1, 2 A variety of reagents, both synthetic and
natural, are capable of interacting with DNA and (or) RNA made up off
purines and pyrimidines (Figure 4.1a and 4.1b). These interactions
may inactivate or completely destroy the nucleic acids. This means the
unwanted or disease causing genes, responsible for the production of
mutated proteins and ultimately leading to the production of
dysfunctional proteins, can be repaired by utilizing the specific
hydrogen bonding patterns between the base pairs. Specially designed
short nucleic acid sequences or oligonucleotides can selectively bind to
the targeted DNA or RNA.
102
Oligonucleotides that selectively bind to the DNA are termed
‘antigene oligonucleotides’3 and the oligonucleotides that selectively
bind to the RNA are called ‘antisense oligonucleotides’4. Peptide
Nucleic Acids are hybrid structures with some special advantages.
Figure 4.1a. DNA double helix structure-sugar phosphate backbone-adenine-thymine, guanine-cytosine bases [courtesy: dearbornschools.org]
Since the discovery of Peptide Nucleic Acids (PNAs) by Nielsen et
al5-6 in 1991 they have attracted significant attention as promising
candidates for the gene therapeutic7-11 drug discovery. PNAs have
applications in therapeutic drug discovery, diagnosis12 and
biosensing13.
103
Figure 4.1b Purines and pyrimidines of nucleic acids, DNA and RNA
PNAs are achiral, neutral, unnatural DNA analogs (Figure 4.2) in
which the nucleobases are attached to the pseudo peptide backbone,
made of repeated units of 2-aminoethylene glycine moieties, via
methylenecarbonyl linkers. PNA’s are interesting pseudo peptides
because of their remarkable affinity and specificity to hybridize with
complimentary nucleic acids, and their resistance to chemical and
biological reactions catalyzed by proteases and nucleases. These
properties are due to their uncharged and flexible polyamide
backbone. They mimic DNA and/or RNA by forming hetero duplexes
with complimentary nucleic acids. The complexes formed by the
interaction of PNA with DNA and/or RNA generally show higher thermal
stabilities than the complexes formed by the interaction of DNA with
DNA or RNA.
104
Figure 4.2. Structures of achiral PNA backbone and chiral DNA backbone
The PNAs bind to the complimentary DNA and RNA sequences
through Watson-Crick, and Hoogsteen in some cases, hydrogen
bonding with higher affinity than the corresponding DNA or RNA
sequences.
Even though the peptide nucleic acids are considered as
promising candidates for the gene therapeutic drug discovery due to
their remarkable stability towards nucleases and proteases and have
many desirable properties, there are some drawbacks which have to
be answered in order to have these macromolecules ready as drug
candidates. Current PNAs exhibit poor cellular uptake14-15 in vitro and
poor bioavailability in vivo. This inherent limitation has prevented
105
widespread application of PNAs as therapeutics as well as in basic
research. Unmodified PNA oligomers are essentially not taken up by
eukaryotic or prokaryotic cells in vitro and when delivered in animals
in vivo (intravenous or intraperitoneal) they are quickly excreted
through the kidneys. This is partly attributed to the uncharged
property of the peptide nucleic acids. In comparison, Nucleic acid
oligonucleotides are negatively charged phosphates, and can readily
penetrate through the cell membrane when complexed with positively
charged lipids. To solve this problem some researchers synthesized
positively charged PNAs based on lysine and arginine residues16-19.
Inspired by the idea to increase cellular uptake by introducing the
positively charged species into the PNA, we were interested in
investigating the cellular uptake of cysteine based PNAs abbreviated as
CPNAs.
Figure 4.3. Proposed structure of cysteine based PNA (CPNA)
106
The reason behind selecting cysteine is the presence of thiol
group. This gives us an easy entry to synthesize more varied PNAs by
using a variety of alkyl groups from a simple methyl group to the long
poly-ether side chain as well as the guanidine-based side chain shown
in Figure 4.3.
4.2 Results and discussion:
4.2.1 Synthesis of standard PNA monomers:-
The strategy of PNA synthesis is based on solid phase chemistry.
For this strategy to work, protected PNA monomers were synthesized
based on the ethylene diamine precursor.20 The monomers were
synthesized following the standard protocols in the literature. Fmoc-
based monomers are especially needed to monitor the oligomerization
using UV-based probes.21
Readily available ethylene diamine is mono protected with di-t-
butyl carbonate anhydride to 4.1 and further protection of 1o amine
group with Fmoc-Cl yielded 4.2 in very good yields. Deprotection of
the Boc group and subsequent alkylation with tert-butyl bromo acetate
gave mono alkylated amine 4.4 as major product in very good yields.
107
Submonomer 4.4 is coupled with orthogonally protected nucleobase
acetic acids to get compounds 4.5a-d. Deprotection of tertiary butyl
ester in mild acid conditions gave desired monomers 4.6a-d in
excellent yields. (Scheme 4.1)22-24
Scheme 4.1 Synthesis of Fmoc protected PNA monomers
Synthesis of PNA monomers for Boc based solid phase synthesis
are started with the common intermediate 4.1. In this scheme,
compound 4.1 is reacted with methyl bromo acetate to give
submonomer 4.7 in very decent yields which is reacted with
orthogonally protected nucleobase acetic acids using uranium complex
HATU as coupling agent to get compounds 4.8a-d in very good yields.
(Scheme 4.2).25
108
Scheme 4.2 Synthesis of Boc protected PNA monomers
4.2.2 Synthesis of CPNA monomers:
Cysteine-based PNA monomers are synthesized starting from the
commercially readily available S-Trityl-N-Boc L-cysteine. (Scheme 4.3)
Synthesis involves the activation of carboxylic acid with DCC followed
by attack of amine nucleophile to enable the formation of amide
4.1026. Upon selective reduction of amide 4.10 with etherated borane
yielded amine 4.11 in very good yields.27 Alkylation of the amine via
SN2 reaction using DIEA as base gave the desired mono alkylated ester
4.12 in moderate yields. Selective unmasking of thiol group and
subsequent alkylation with methyl iodide gave submonomer 4.14 in
109
decent yields.28 The coupling of orthogonally protected nucleobase
acetic acids to the submonomer 4.14 gave corresponding methyl
esters 4.15a-d in pretty good yields. Methyl esters were hydrolyzed in
alkaline conditions to get CPNA monomers 4.16a-d in very good
yields.29
Scheme 4.3 Synthesis of novel cysteine PNA monomers
4.2.3 Synthesis of novel polyether side chain:
Oligo-ethers are known to have water solubility compared to the
alkyl analogues because of the presence of oxygen group which can
form hydrogen bonds with the water in biological systems.30-31 This
property encouraged us to come up with a new alkyl group 4.21
110
(Scheme 4.4) for our ongoing PNA synthesis. Synthesis of the
compound 4.21 starts with the commercially available 3-
bromopropanamine salt. 3-bromopropanamine hydrobromide salt was
reacted with di-t-butyl dicarbonate in aqueous medium in the presence
of sodium bicarbonate to afford quantitative yields of 4.17. Excess
sodium bromide was used to make sure the bromide is not hydrolyzed
to the corresponding hydroxide. Bromide 4.17 was alkylated onto
triethylene glycol monomethyl ether 4.18 using sodium hydride to get
4.19 in decent yields. Deprotection of the Boc group and subsequent
reaction with bromo acetyl bromide gave 92% of the amide 4.21.32
Scheme 4.4 Synthesis of novel polyether side chain
111
Novel side chain 4.21 can be alkylated onto 4.13 to achieve
novel cysteine based monomers and further achieve PNAs with novel
properties as to achieve the goals of this project.
4.3 Solid phase synthesis of PNAs
Standard solid phase synthesis protocol was followed for the
synthesis of PNA oligomers for both normal as well as cysteine based
PNA synthesis. The Symphony instrument from Protein Technologies
Inc was used for the synthesis of Fmoc based methods on MBHA resin.
Manual synthesis used the Boc strategy. MBHA resin was swollen in
NMP for 45 minutes and was downloaded with Fmoc-Lys(Boc)-OH
before growing PNA chain. Substitution level of approximately 0.15
mmol/g was maintained so as to achieve the best results as reported
in the literature. Fmoc deprotection was then performed using
piperidine (20%) and DBU (2%) in NMP. Coupling of monomers with
protecting group was carried out with excess of reagents (at least five
fold excess). After the coupling is complete the resin was washed with
necessary solvents and the same cycle continued until the desired
length and sequence was achieved. After the final coupling cycle PNA
was global deprotected and cleaved from the resin using TFA, DMS and
TFMSA
112
4.4. Solid phase synthesis results
Table 4.1. Solid phase synthesis of PNAs
4.5 Conclusion
Syntheses of standard PNA monomers and novel Cysteine PNA
monomers were successfully achieved. Optimizations were done to
synthesize newly designed CPNA monomers with varying alkyl groups
on sulphur group. Partially successful results were tabulated above and
the schemes mentioned and developed for the synthesis of monomers
will be utilized in the future synthesis of PNAs.
113
4.6 Experimental procedures
(2-Amino-ethyl)-carbamic acid tert-butyl ester (4.1): To the ice
cold solution of ethylene diamine (13.48 g, 224.3 mmol) in 250 mL of
tetrahydrofuran was added a solution of di-t-butyl carbonate anhydride
(10.0 g, 46 mmol) in 50 mL of tetrahydrofuran over a period of 25
minutes. The reaction was continued at 0 oC for 3 hours and then
brought to ambient temperatures and continued for 20 more hours.
Upon complete consumption of the di-t-butyl carbonate anhydride, the
reaction mixture was concentrated under reduced pressure. The
residue obtained was dissolved in ethyl acetate (150 mL) and washed
with water (4×45 mL). The aqueous layer was back extracted with
ethyl acetate; organic portions were mixed, dried over anhydrous
sodium sulphate (Na2SO4), and concentrated under reduced pressure.
The crude product was flash chromatographed to give mono Boc
protected amine 4.1 in good quantities, 5.8 g (79%). 1H NMR (DPX
250 MHz, CDCl3) δ 5.35 (bs, 1H) 3.16 (m, J=5.75, 2H), 2.78 (t, J=5.5,
2H), 1.45 (s, 9H), 1.3 (s, 2H). 13C NMR (DPX 250 MHz, CDCl3) δ
114
156.3, 78.9, 43.2, 41.7, 28.3. LCMS (ESI) m/z calcd for C7H16N2O2 is
160.1, found 161.2 [M+H]+
tert-Butyl N-[2-(N-9-fluorenylmethoxycarbonyl) amino ethyl]
glycinate (4.2): To the ice cold solution of (2-amino-ethyl)-carbamic
acid tert-butyl ester 4.1 (0.5 g, 3.120 mmol) in 100 mL of dry
dichloromethane was added diisopropylethylamine (0.443 g, 3.43
mmol). To the resulting solution of 9-fluorenylmethyl chloroformate in
7 mL of dichloromethane was added over a period of 15 minutes. The
reaction was stirred at 0 oC for two hours. After the reaction was
complete, the same was diluted to 150 mL with dichloromethane and
washed with 45 mL of half saturated brine solution. The organic layer
was dried over anhydrous Na2SO4 and concentrated. The crude
product was purified by precipitation using ethyl acetate and hexanes
to get 1.19 g (90.75%) of 4.2 as a white fluffy solid. 1H NMR (DPX 250
MHz, CDCl3) δ 7.78-7.72 (d, 2H, J=7.5); 7.62-7.56 (d, 2H, J=7.5);
7.44-7.24 (m, 4H); 5.22 (bs, 1H); 4.82 (bs, 1H); 4.42 (d, 2H,
J=6.75); 4.21 (t, 1H, J=5.5); 3.28 (bs, 4H); 1.44 (s, 9H).
115
2-(9H-Fluoren-9-ylmethoxycarbonylamino)-ethyl-ammonium
chloride (4.3):- A solution of orthogonally protected ethylene
diamine 4.2 (0.5 g, 1.307 mmol) in 15 mL of 1, 4-dioxane was cooled
to 0 oC. HCl gas was passed through the solution for 4-5 minutes and
reaction was brought to ambient temperature by removing the ice
bath. The reaction was monitored by TLC for the progress. After
complete consumption of the starting material, in about 4 hrs, solvent
was removed under reduced pressure and the product was used in the
next step after drying under high vacuum to give pure product 4.3 as
white solid (~ quantitative yields). 1H NMR (DPX 250 MHz CDCl3) δ
7.84 (d, 2H, Fmoc Ar CH); 7.65 (d, 2H, Fmoc Ar CH); 7.44-7.27 m,
5H, Fmoc Ar CH, NHFmoc); 4.42 (d, 2H, J=6.5, Fmoc CH2); 4.22 (t,
1H, J=6.5, Fmoc, CH2); 3.37 (t, 2H, CH2-NHFmoc); 3.30 (bs, 3H,
NH3); 3.02 (t, 2H, CH2-NH3).
116
tert-Butyl N-[2-(N-9-fluorenylmethoxycarbonyl) aminoethyl]
glycinate ester (4.4):-To a cold solution of 4.3 (0.5 g, 1.3 mmol) in
THF (30 mL) were added diisopropylethylamine (0.432 ml, 2.6146
mmol), tetrabutylammonium iodide (0.155 g, 0.156 mmol) and tert-
butyl bromoacetate (0.195 ml, 1.3073 mmol) under stirring. Reaction
was continued for 10 hrs at 0 oC and then brought to ambient
temperature. Reaction mixture was concentrated under reduced
pressure and flash chromatographed to get the desired mono alkylated
product 4.4 as white semi-solid (0.181 g, 36%). 1H NMR (DPX 250
MHz CDCl3) δ 7.67 (d, 2H, J=7.25, Fmoc Ar CH); 7.52 (d, 2H, J=7.25,
Fmoc Ar CH); 7.35-7.16 (m, 5H, Fmoc Ar CH, NH); 5.39 (bs, 1H, NH);
4.31 (d, 2H, J=6.75, Fmoc CH2); 4.13 (t, 1H, J=6.75, Fmoc CHO);
3.21 (bs, 1H, NH); 2.67(t, 2H, CH2) 1.92 (s, 2H, NHCH2CO); 1.40 (s,
9H, tBu).
General procedure for nucleo-base coupling (4.5a-d):- DIEA was
added to a solution of Cbz protected nucleo-base acetic acid, HATU
and compound 4.4 in DMF at room temperature. The reaction was
monitored for its progress. Starting material was completely consumed
within 20–25 minutes. Solvent was removed under high vacuum and
the residue was dissolved in ethyl acetate and washed with saturated
brine solution. The organic layer was dried over anhydrous Na2SO4 and
117
concentrated. Flash column chromatography of the crude gave coupled
products 4.5a-4.5d in 65-95% yield.
{[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-ethyl]-[2-(5-
methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-acetyl]-
amino}-acetic acid tert-butyl ester (4.5a) white solid, 96% yield.
1H NMR (DPX 250 MHz, DMSO-d6) δ 7.89 (d, 2H, J=7.5 Fmoc Ar CH);
7.68 (d, 2H, J=7.5, Fmoc Ar CH); 7.46-7.23 (m, 5H, Fmoc Ar CH, NH);
4.64 (s, 1H, CH); 4.45 (s, 1H); 4.32 (d, 2H, J=, Fmoc CH2); 4.22 (t,
1H, J=, Fmoc CH), 1.73 (s, 3H, CH3); 1.45-1.39 (ss, 9H, -C(CH3)3).
13C NMR (DPX 250 MHz, DMSO-d6) δ 167.9, 167.2, 164.3, 156.2,
150.9, 143.8, 142.1, 140.7, 127.6, 127.0, 125.1, 120.1, 108.1, 81.9,
80.9, 48.7, 47.6, 46.9, 27.6.
118
4.5b
{[2-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-
acetyl]-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl]-
amino}-acetic acid tert-butyl ester (4.5b) white slid, 94% yield.
1H NMR (DPX 250 MHz, CDCl3) δ 10.81 (s, 1H), 7.92 (m, 3H), 7.75 (d,
2H), 7.62-7.21 (m, 9H), 7.02 (d, 1H), 5.23 (s, 2H), 4.81 (s, 1H), 4.66
(s, 1H), 4.44-4.25 (m, 4H), 3.93 (s, 2H), 3.46 (2H), 3.1 (2H), 1.42
(s, 9H).
{[2-(5-Benzyloxycarbonylamino-7-oxo-6,7-dihydro-
[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-acetyl]-[2-(9H-fluoren-9-
ylmethoxycarbonylamino)-ethyl]-amino}-acetic acid tert-butyl
119
ester (4.5c) white solid, 82% yield. 1H NMR (DPX 250 MHz, MeOD-d4)
δ 11.35 (1H, NH); 7.87(d, 2H); 7.81 (d, 1H); 7.43-7.34 (m, 9H); 5.24
(3H); 5.08 (s, 1H); 4.43-4.18 (m, 4H); 3.96 (s, 1H); 1.53-1.34 (s,
9H).
{[2-(6-Benzyloxycarbonylamino-purin-9-yl)-acetyl]-[2-(9H-
fluoren-9-ylmethoxycarbonylamino)-ethyl]-amino}-acetic acid
tert-butyl ester (4.5d) white solid, 87% yield. 1H NMR (DPX 250
MHz, MeOD-d4) δ 10.67 (s, 1H, NH); 8.55 (s, 1H); 8.32 (s, 1H); 7.89
(d, 2H, J=7.5); 7.68 (m, 2H); 7.51-7.21(m, 12H); 5.35 (s, 1H); 5.25
(s, 1H); 4.42-4.25 (m, 4H); 4.12-3.94(m, 2H); 3.64 (s, 1H); 2.02 (s,
2H); 1.53-1.35 (9H).
General procedure for the deprotection of t-butyl esters 4.5a-d:
120
To a stirred solution of ester 4.5 in dichloromethane was added TFA at
ice cold temperatures. After the completion of deprotection TFA was
removed under reduced pressure. The residue was dissolved in DCM
and subjected to rotary evaporation and the same was repeated 3-5
times so as to remove the TFA to the maximum. And the crude residue
was subjected to flash column chromatography to get pure compounds
4.6a-d as solids.
{[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-ethyl]-[2-(5-
methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-acetyl]-
amino}-acetic acid (4.6a) white solid, 92% yield. 1H NMR (DPX 250
MHz, DMSO-d6) δ 11.02 (s, 1H), 7.35 (1H), 7.02 (1H), 4.76 (s, 2H),
121
4.57 (1H), 4.33 (s, 1H), 4.02 (2H), 3.74 (s, 1H), 3.63 (s, 3H), 3.44-
3.03 (m, 3H), 1.75 (s, 3H), 1.4 (s, 9H).
{[2-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-
acetyl]-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl]-
amino}-acetic acid (4.6b) white solid, 87% yield. 1H NMR (DPX 250
MHz, DMSO-d6) δ 10.93 (bs, NH, 1H), 7.94 (1H), 7.55-7.44 (5H), 7.0
(1H), 5.23 (s, 2H), 4.82 (s, 2H), 4.12 (s, 2H), 3.65 (s, 3H), 3.45-2.93
(m, 4H), 1.42 (s, 9H).
122
{[2-(5-Benzyloxycarbonylamino-7-oxo-6,7-dihydro-
[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-acetyl]-[2-(9H-fluoren-9-
ylmethoxycarbonylamino)-ethyl]-amino}-acetic acid (4.6c)
yellowish solid, 86% yield. LCMS (ESI) m/z calcd for C24H29N7O7 is
527.2, found 528.1 [M+H]+.
{[2-(6-Benzyloxycarbonylamino-purin-9-yl)-acetyl]-[2-(9H-
fluoren-9-ylmethoxycarbonylamino)-ethyl]-amino}-acetic acid
(4.6d) white solid, 84% yield. 1H NMR (Inova 400 MHz, DMSO-d6) δ
8.56 (d, J = 19.3 Hz, 1H), 8.49–8.45 (m, 1H), 7.84 (d, J = 7.5 Hz,
2H), 7.67–7.61 (m, 2H), 7.50–7.46 (m, 1H), 7.45–7.42 (m, 2H),
7.39–7.34 (m, 4H), 7.34–7.30 (m, 1H), 7.29–7.25 (m, 2H), 5.35 (s,
1H),5.19 (d, J = 18.2 Hz, 3H), 4.33 (d, J = 7.1 Hz, 2H), 4.28–4.14
(m, 3H), 3.97 (s, 1H),3.55–3.50 (m, 1H), 3.33 (d, J = 15.4 Hz, 2H).
123
Methyl N-(2-Boc-aminoethyl) glycinate (4.7):-
Diisopropylethylamine (DIEA) (2.47 ml, 15.0 mmol) was added to an
ice cold solution of (2-amino-ethyl)-carbamic acid tert-butyl ester 4.1
(2.0 g, 12.5 mmol) in 100 ml of dry tetrahydrofuran at 0 oC under
nitrogen atmosphere. To the above solution, methyl bromoacetate
(1.18 ml, 12.5 mmol) was added over a period of 15 minutes followed
by the addition of tetra butyl ammonium iodide, TBAI (0.23 g, 0.6245
mmol). The temperature is maintained near 0 oC for 9 hrs and then
brought to ambient temperature and continued for another 10 hours.
Solvent is stripped off under reduced pressure and the crude is flash
column chromatographed to get (2.1 g, 72%) of the mono alkylated
compound 4.7 along with unwanted di alkylated compound. 1H NMR
(DPX 250 MHz, CDCl3) δ 5.13 (bs, 1H, NH); 3.75 (s, 3H, CH3); 3.44 (s,
2H, CH2); 3.23 (q, 2H, CH2, J=5.75); 2.73 (t, 2H, CH2, J=5.5); 2.18
(bs, 1H, NH); 1.44 (s, 9H);
124
Same procedure was followed for coupling of 4.7 with nucleobase
acetic acids to make 4.8a-d as mentioned in the synthesis of 4.5a-d.
{(2-tert-Butoxycarbonylamino-ethyl)-[2-(5-methyl-2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-yl)-acetyl]-amino}-acetic acid
methyl ester (4.8a) white solid, 93% yield. 1H NMR (DPX 250 MHz,
DMSO-d6), δ 11.0 (s, 1H), 7.3 (1H), 7.0 (1H), 4.7 (1H), 4.1 (1H), 3.7
(1H), 3.6 (s, 3H), 3.5-3.0 (m, 5H), 1.8 (s, 3H), 1.4 (s, 9H)
[[2-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-
acetyl]-(2-tert-butoxycarbonylamino-ethyl)-amino]-acetic acid
methyl ester (4.8b) white solid, 91% yield. 1H NMR (DPX 250 MHz,
MeOD-d4) δ 10.8 (s, 1H), 7.8 (d, 1H), 7.4 (m, 5H), 7.0 (d, 1H), 5.2 (s,
2H), 4.6 (s, 1H), 4.5-3.5 (m, 6H), 3.4 (s, 3H), 2.5 (s, 2H), 1.4 (s, 9H).
125
[[2-(5-Benzyloxycarbonylamino-7-oxo-6,7-dihydro-
[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-acetyl]-(2-tert-
butoxycarbonylamino-ethyl)-amino]-acetic acid methyl ester
(4.8c) white solid, 77% yield. 1H NMR (DPX 250 MHz, DMSO-d6), δ
11.5 (s, 1H), 11.4 (s, 1H), 7.8 (s, 1H) 7.6-7.3 (m, 5H), 5.3 (s, 2H),
4.1-3.2 (m, 11H), 1.4 (s, 9H)
[[2-(6-Benzyloxycarbonylamino-purin-9-yl)-acetyl]-(2-tert-
butoxycarbonylamino-ethyl)-amino]-acetic acid methyl ester
(4.8d) white solid, 86% yield. 1H NMR (DPX 250 MHz, DMSO-d6) δ
10.7 (s, 1H), 8.5 (s, 1H), 8.2 (s, 1H), 8.0 (s, 1H), 7.8-7.2 (m, 5H), 5.2
(s, 2H), 4.5-4.0 (m, 4H), 3.3 (s, 3H), 2.5 (s, 2H), 1.4 (s, 9H).
126
General procedure for the deprotection of methyl esters 4.8a-d:
To a stirred solution of ester 4.8a-d in THF/H2O was added 1M NaOH in
water at ice cold temperatures. After the completion of deprotection
THF was removed under reduced pressure. The water layer was
extracted with EtOAc to remove any impurities and the water layer
was acidified to ρH 4.0 and the product was extracted into EtOAc and
the organic solvent was dried over anhydrous Na2SO4 and removed
under reduced pressure. The crude product was chromatographed if
necessary to obtain pure products 4.9a-d as solids.
{(2-tert-Butoxycarbonylamino-ethyl)-[2-(5-methyl-2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-yl)-acetyl]-amino}-acetic acid
(4.9a) white solid, 94% yield. 1H-NMR (DPX 250 MHz, DMSO-d6)12.1
127
(s, 1H), 10.2 (bs, 2H), 7.1 (1H), 4.4 (m, 4H), 3.5-3.2 (m, 4H), 1.9
(2H), 1.4 (9H). LCMS (ESI) m/z 285.1 [M+H]+.
[[2-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-
acetyl]-(2-tert-butoxycarbonylamino-ethyl)-amino]-acetic acid
(4.9b) white solid, 93% yield. 1H NMR (DPX 250 MHz, DMSO-d6), δ
11.0 (bs, 2H), 7.9 (m, 1H), 7.5-7.3 (m, 5H), 7.1-6.9 (m, 1H), 5.2 (s,
2H), 4.8 (s, 2H), 4.0 (s, 2H), 3.3-3.1(m, 4H), 1.4 (s, 9H), LCMS (ESI)
m/z 504.2 [M+H]+.
[[2-(5-Benzyloxycarbonylamino-7-oxo-6,7-dihydro-
[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-acetyl]-(2-tert-
butoxycarbonylamino-ethyl)-amino]-acetic acid (4.9c) white
128
solid, 94% yield. 1H-NMR (DPX 250 MHz, DMSO-d6), δ 11.0 (1H), 7.8
(s, 1H), 7.5-7.3 (m, 5H), 5.2 (s, 2H), 4.0 (s, 2H), 3.9-3.7 (m, 2H),
3.6-3.2 (m, 4H), 1.4 (s, 9H); LCMS (ESI) m/z 544.2 [M+H]+.
[[2-(6-Benzyloxycarbonylamino-purin-9-yl)-acetyl]-(2-tert-
butoxycarbonylamino-ethyl)-amino]-acetic acid (4.9d) white
solid, 82% yield. LCMS (ESI) m/z calcd for C24H29N7O7 is 527.21, found
528.1 [M+H]+.
(R)-tert-butyl 1-amino-1-oxo-(tritylthio) propan-2-ylcarbamate
(4.10):- To a solution of commercially available S-trityl Boc Cysteine
(2.0 g. 4.31 mmol), HOBt (0.70 g, 5.17 mmol) and DCC (1.07 g, 5.18
mmol) in THF (10ml) was added 28 % NH4OH (0.44 ml, 6.46 mmol) at
129
0 oC. After 2 hr of stirring at 0 oC, the reaction mixture was filtered
through Celite and filtrate was concentrated, diluted with ethyl
acetate, and washed with water and brine. Organic layer was then
dried over anhydrous sodium sulphate and concentrated under
reduced pressure. The crude is flash chromatographed to get pure
compound 4.10 as a white solid in nearly quantitative yields. (1.95 g,
98%) 1H NMR (DPX 250 MHz, CDCl3) δ 7.54-7.24 (15H, C6H5); 6.03
(bs, 1H); 5.55 (bs, 1H); 4.87 (bs, 1H); 3.93 (bs, 1H); 2.02 (s, 2H,
CH2) 1.44 (s, 9H); 13C NMR (DPX 250 MHz, CDCl3) δ 173.83, 155.42,
144.42, 130.04, 129.60, 128.10, 126.93, 80.34, 67.24, 53.22, 33.80,
28.33.; LCMS (ESI) m/z calcd for C27H30N2O3S is 462.19, found 485.16
[M+Na]+.
(R)-tert-butyl-1-amino-3(tritylthio) propan-2-ylcarbamate
(4.11): - To the solution of 4.10 (1 g, 2.16 mmol) in anhydrous THF
(5 ml) was added 1M Borane-THF complex (5.4 ml, 5.4 mmol) at 0 oC
and stirred at 0 oC for 3 hrs. The reaction was heated to 60 oC and
refluxed for 12 hrs. The reaction mixture was cooled to 0 oC and
130
quenched by slow addition of methanol. The oil obtained upon
repeated dilutions and concentrations (2-3 times) with methanol, was
subjected to water work up (pH 8.5) followed by flash column
chromatography to afford the corresponding amine 4.11 (0.58 mg, 60
%). 1H NMR (DPX 250 MHz, CDCl3) δ 7.40 (D, J=7.27 Hz, 6H), 7.19
(ddd, J=14.29, 5.76 Hz, 9H), 5.27-4.85 (bs, 1H), 3.55 (bs, 2H), 2.35
(tt, J=17.88, 8.99 Hz, 2H), 2.63 (d, J=5.68 Hz, 2H), 1.39 (s, 3H). 13C
NMR (DPX 250 MHz, CDCl3) δ 155.57, 144.65, 79.35, 66.79, 64.35,
60.39, 44.40, 34.37.LCMS (ESI) m/z calcd for C27H32N2O2S is 448.21,
found 449.22 [M+H]+.
(2-tert-Butoxycarbonylamino-3-tritylsulfanyl-propylamino)-
acetic acid methyl Ester (4.12):-To the solution of 4.11 (1.66 g,
3.7 mmol) in THF (40 ml) at 0 oC was added methyl bromoacetate (35
ml, 3.7 mmol, dissolved in 5 ml) followed by the addition of DIEA
(0.672 ml, 4.070 mmol) and stirring was continued for 7 hrs. Reaction
was brought to ambient temperature and continued for another 10
hrs, after which it was concentrated to dryness. The residue obtained
was dissolved in ethyl acetate and washed with saturated brine
131
solution. Aqueous layer was back extracted into ethyl acetate. Organic
layer was dried over anhydrous sodium sulfate and concentrated to
give crude product which was purified by flash column
chromatographed to get pure compound 4.12 (0.721 g, 37.43 %). 1H
NMR (DPX 250 MHz, CDCl3) δ 7.54-7.12 (m, Ar 15H); 4.74 (bs, NH,
1H); 3.73 (s, CH3, 3H);3.33 (d, NHCH2CO, 2H); 2.72-2.26 (m, 3H)
1.64 (s, SCH2, 2H); 1.44 (s, tBu, 9H). 13C NMR (DPX 250 MHz, CDCl3)
δ 172.8, 155.6, 79.48, 51.8, 51.2, 50.8, 49.7, 36.9, 28.4, 16.3, HRMS
(ESI) m/z calcd for C30H36N2O4S is 520.24, found 521.2 [M+H]+.
(2-tert-Butoxycarbonylamino-3-methylsulfanyl-propylamino)-
acetic acid methyl ester (4.14):-To the solution of compound 4.12
(0.700, 1.34 mmol) in 100 ml of dry dichloromethane was added TFA
(1.6 ml) at 0 oC over a period of 7 minutes which turned into yellow
color. To the above solution, triethylsilane (0.436 ml) was added over
a period of 3 minutes. The reaction mixture turned to a pale yellow
from dark yellow color. Reaction was complete in 12 minutes by TLC.
Reaction mixture was concentrated to half of its volume and diluted
132
with 50 ml of DCM. Again concentrated and repeated the same thing
for four times to make sure TFA was removed as much as possible.
The crude compound 4.13 was dried under high vacuum and taken to
the next step without further purification. The above residue was
dissolved in 10 ml of Methanol and cooled to 0 oC to which 3.36 ml of
1N NaOH was added followed by the addition of 0.16 ml of
iodomethane. The reaction was over in 15 minutes. The reaction
mixture was neutralized by adding 20% citric acid solution and the
solvent was removed under vacuum. The residue was dissolved in
ethyl acetate and washed with saturated brine solution. The organic
layer was dried over anhydrous sodium sulphate and concentrated.
Flash chromatography gave pure compound 4.14 (0.22 g, 56%). 1H
NMR (DPX 250 MHz, CDCl3) δ 3.73 (s, CH3, 3H); 3.53 (d, 2H); 2.91-
2.62 (m, 3H); 2.38 (bs, 2H); 2.25 (s, 3H); 1.93 (s, tBu, 9H); LCMS
(ESI) m/z calcd for C12H24N2O4S is 292.14, found 293.1 [M+H]+.
Representative base coupling procedure: Diisopropylethylamine
(0.37 ml, 0.29 mmol) was added to a stirring solution of 4.14 (0.22 g,
133
0.75 mmol), thymine acetic acid (0.207 g, 1.1278 mmol) and HATU
(0.343g, 0.90 mmol) in DMF (3 ml) at ambient temperatures. The
reaction mixture turned yellow. TLC showed the reaction was complete
in 10 minutes. DMF was removed under high vacuum without heating.
The crude was dissolved in ethyl acetate (150 ml) and washed with
saturated brine (7 ml) The layers were separated and the organic layer
was dried over anhydrous sodium sulphate and concentrated. Flash
chromatography of the crude gave pure compound 4.15a as white
solid (0.29 g, 87.8%). The same procedure is used to synthesize
4.15a-d compounds.
4.15a: product as white solid, 0.29 g, 87.8%. 1H NMR (DPX 250 MHz,
CDCl3) δ 7.33 (1H), 4.42 (s, 1H), 3.85 (s, 2H), 4.34-3.96 (m, 2H),
3.74 (s, 3H), 3.33 (1H), 2.74-2.55 (m, 2H), 2.27 (s, 2H), 2.13 (2H),
1.93 (s, 3H), 1.53 (s, tBu, 9H); LCMS (ESI) m/z calcd for C19H30N4O7S
is 458.18, found 458.19 [M+H]+, 481.17 [M+Na]+.
134
4.15b: pure product as white solid, (0.168 g, 85.27%).1H NMR (DPX
250 MHz, CDCl3) δ 8.71(d, NH), 8.35 (NH), 7.85 (d, CH), 7.56-7.23
(Ar, 5H),4.7( 1H), 4.41 (d, CH), 4.16 (CH2), 3.80 (CH2), 3.75 (CH2),
3.70 (CH2), 2.21 (CH2), 2.02 (CH3), 1.45 (tBu); LCMS (ESI) m/z calcd
for C26H35N5O8S is 577.22, found 578.2 [M+H]+.
4.15c: (0.33 g, 56.21%); 1H NMR (DPX 250 MHz, MeOD-d4) δ 8.8 (s,
1H), 8.4 (1H), 7.6-7.2 (m, 5H), 5.5 (1H), 5.3 (1H), 4.0-3.6 (4H), 3.4-
3.1 (m, 2H), 3.0 (2H), 2.9 (3H), 1.4 (s, 9H); LCMS (ESI) m/z calcd for
C27H35N7O8S is 617.22, found.618.2 [M+H]+.
135
Hydrolysis of methyl esters was performed following the same
procedure employed for the hydrolysis of 4.8a-d to get pure
compounds 4.16a-d.
(4.16d): colorless solid, 84% yield. 1H-NMR (DPX 250 MHz, MeOD-d4)
δ 8.8 (1H), 8.6 (1H), 8.0 (2H), 7.6-7.3 (3H), 5.5 (2H), 5.3 (2H), 4.3-
3.6 (5H), 3.0 (2H), 2.8 (2H), 2.2 (3H), 1.4 (9H)
136
N-tert-butoxycarbonyl-3-bromopropylamine (4.17): - A solution
of sodium bicarbonate (0.39 g, 4.64 mmol) in 15 ml of water was
added to 3-bromopropylamine hydrobromide (1.0 g, 4.56 mmol) in 20
ml of chloroform. To the above mixture, di tert-butyl dicarbonate
anhydride (1.0 g, 4.58 mmol) was added at ambient temperature, with
vigorous stirring, followed by the addition of sodium bromide (1.0 g,
9.72 mmol). The reaction mixture was refluxed for 18 hrs and then
brought to ambient temperature and the layers were separated. The
aqueous layer was extracted with chloroform (2x20 ml). The organic
layers were mixed and dried over anhydrous sodium sulphate and
concentrated to give pure compound 4.17 without any further
purification (1.04 g, ~100%) 1H NMR (DPX 250 MHz, CDCl3) δ 4.71
(bs, 1H, NH); 3.44 (t, 2H, J=6.5); 3.27 (q, 2H, J=6.5); 2.05 (m, 2H,
J= 6.5); 1.44 (s, 9H, CH3);
3-(2-(2-(2-Methoxy-ethoxy)-ethoxy)-ethoxy)-propyl)-carbamic
acid tert-butyl ester (4.19): - Sodium hydride (60% in mineral oil,
58 mg, 1.461 mmol) was added to an ice cold solution of triethylene
137
glycol monomethyl ether 4.18 (0.2 g, 1.218 mmol) in tetrahydrofuran
(15 ml) and stirred for five minutes. N-tert-Butoxycarbonyl-3-
bromopropylamine 4.17 (0.29 g, 1.218 mmol) was added to the above
mixture at the same temperature. The reaction was gradually brought
to ambient temperature and stirred for another 8 hrs. The reaction
mixture was then cooled to 0 oC and quenched by slow addition of
methanol (10 ml) and concentrated to dryness. The residue was
dissolved in ethyl acetate and washed with saturated brine solution.
The organic layer was dried over anhydrous sodium sulphate and
concentrated. Flash column chromatography yielded the pure
compound 4.19 (0.20 g, 52%). 1H NMR (DPX 250 MHz, CDCl3) δ 5.14
(bs, 1H, NH); 3.73-3.54 (m, 16H) 3.42 (s, 3H, CH3); 3.26 (q, 2H,
J=6); 1.76 (p, 2H, J=6.25, 6.0); 1.44 (s, 9H)
3-{2-[2-(2-Methoxy ethoxy) ethoxy-]-ethoxy}-propylamine
hydrochloride (4.20): - Dry HCl gas was passed into an ice cold
solution of compound 4.19 (0.179 g, 0.55 mmol) dissolved in 1,4-
dioxane (25 ml) for five minutes. Progress of the reaction was
138
monitored by TLC. The reaction was complete in 25 minutes. The
solvent was removed under reduced pressure and dried under high
vacuum. The compound 4.20 was used in the next step without any
further purification.
3-{2-[2-(2-Methoxy ethoxy) ethoxy-]-ethoxy}-propylamine
bromo acetamide (4.21):- The above hydrochloride salt (4.20) is
dissolved in benzene (20 ml) and cooled to 0 oC. To the solution, was
added potassium carbonate (0.153 g, 1.114 mmol) followed by the
addition of bromo acetyl bromide (0.11 g, 0.55 mmol). The reaction
was brought to ambient temperature and stirred for 8 hrs. TLC showed
there was no starting material left. Solids were filtered and the
solution was concentrated under reduced pressure. The residue was
dissolved in ethyl acetate and washed with water and saturated brine
solution. Layers were separated and the organic layer was dried over
anhydrous sodium sulphate and concentrated under reduced pressure
to give crude which was purified by flash column chromatography to
give the amide compound 4.21. 1H NMR (DPX 250 MHz, CDCl3) δ 9.33
(bs, 1H, NH); 4.34-3.52 (16H); 3.43 (s, 3H, CH3); 2.13 (s, 2H, CH2);
139
13C NMR (250 MHz, CDCl3) δ 171.23, 71.76, 70.41, 70.35, 70.32,
70.25, 69.00, 63.55, 58.84, 38.93, 28.85, 20.85.
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145
146
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APPENDIX-A: Selected 1H, 13C NMR Spectra & Mass spectra
147
2-N-(methanesulfonyl)-aminopyrimidine (2.1)
148
5-iodo-2-N-(methanesulfonyl) amino pyrimidine (2.2)
149
5-iodo-2-N-[(methanesulfonyl), (4-methoxybenzyl)] amino pyrimidine (2.3)
150
5-[(2-hydroxy), (2-methyl)] butynyl-2-N-[(methanesulfonyl), (4-methoxynenzyl)] amino pyrimidine (2.4)
151
N-(5-Ethynyl-pyrimidin-2-yl)-N-(4-methoxy-benzyl)-methanesulfonamide (2.5)
152
N-(5-{2-[Methanesulfonyl-(4-methoxy-benzyl)-amino]-pyrimidin-5-ylethynyl}-pyrimidin-2-yl)-N-(4-methoxy-benzyl)-
methanesulfonamide (2.6)
153
N-{5-[2-(2-Methanesulfonylamino-pyrimidin-5-yl)-ethyl]-pyrimidin-2-yl}-methanesulfonamide (2.8)
154
5-iodo-2-amino-pyrimidine (2.9)
155
Di-tert-butyl (5-iodopyrimidin-2-yl) dicarbamate (2.10)
156
(5-{2-[Methanesulfonyl-(4-methoxy-benzyl)-amino]-pyrimidin-5-ylethynyl}-pyrimidin-2-yl)-di-carbamic acid di-
tert-butyl ester (2.11)
157
1H-NMR, 13C-NMR & 13C-DEPT135 (2.12)
158
159
N-{5-[2-(2-Amino-pyrimidin-5-yl)-ethyl]-pyrimidin-2-yl}-N-(4-methoxy-benzyl)-methanesulfonamide (2.13)
160
C-Chloro-N-[5-(2-{2-[methanesulfonyl-(4-methoxy-benzyl)-amino]-pyrimidin-5-yl}-ethyl)-pyrimidin-2-yl]-
methanesulfonamide (2.14)
161
C-Chloro-N-{5-[2-(2-methanesulfonylamino-pyrimidin-5-yl)-ethyl]-pyrimidin-2-yl}-methanesulfonamide (2.15)
162
2-N-(chloro methanesulfonyl)-aminopyrimidine (2.16)
163
L-Serine methyl ester hydrochloride (2.19)
164
Die )
thyl-(phenylcarbonyl)-phosphonate (3.2
165
Phosphonic acid, (2-phenyloxiranyl)-, diethyl ester (3.3)
166
(2-Phenyl-oxiranyl)-lithium phosphonate mono-ethyl ester (3.4)
167
Diazo-phenyl-acetic acid methyl ester (3.7)
168
2,3-diphenyl-oxirane-2-carboxylic acid methyl ester (3.8a)
169
3-(2- thyl ester (3.8g)
nitro-phenyl)-2-phenyl-oxirane-2-carboxylic acid me
3-(2-nitro-phenyl)-2-phenyl-oxirane-2-carboxylic acid (3.9g)
170
[(Methoxycarbonyl-phenyl-methylene)-hydrazono]-phenyl-id methyl esteacetic ac r (3.8i)
171
2,3-diphenyloxirane (3.10)
172
β-azido-α-phenyl benzeneethanol (3.11)
173
2,3-Diphenylaziridine (3.12)
174
1-Aziridinecarboxylic acid,2,3-diphenyl-, 1,1-dimethylethyl ester (3.13)
175
N-(phenylmethyle thanamine (3.16)ne)-me
176
Be )nzenemethanamine-4-methoxy-N-(phenylmethylene) (3.17
177
Benzyl-benzylidene-amine (3.18)
178
1,2-diphenyl-cyclopropanecarboxylic acid methyl ester (3.21)
179
1,2-diphenyl-cyclopropanecarboxylic acid (3.22)
180
(2-Amino-ethyl)-carbamic acid tert-butyl ester (4.1)
181
tert-Butyl N-[2-(N yl) amino ethyl] glycinate (4.2)
-9-fluorenylmethoxycarbon
182
2-( m chloride (4.3)
9H-Fluoren-9-ylmethoxycarbonylamino)-ethyl-ammoniu
183
tert-Butyl N-[2-(N-9-fluorenylmethoxycarbonyl) aminoethyl] glycinate ester (4.4)
184
(4.5a)
[2-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-acetyl]-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl]-
amino}-acetic acid tert-butyl ester (4.5b)
185
[2-(5-Benzyloxycarbonylamino-7-oxo-6,7-dihydro-idin-3-yl)-
hyl]-a[1,2,3]triazolo[4,5-d]pyrim acetyl]-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-et mino}-acetic acid tert-butyl
ester (4.5c)
186
[2-(6-Benzyloxycarbonylamino-purin-9-yl)-acetyl]-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl]-amino}-acetic acid
tert-butyl ester (4.5d)
187
2-(9H-Fluoren-9-ylmethoxycarbonylamino)-ethyl]-[2-(5--dihydro-2H-pyrmethyl-2,4-dioxo-3,4 imidin-1-yl)-acetyl]-
amino}-acetic acid (4.6a)
188
[2-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-acetyl]-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl]-
amino}-acetic acid (4.6b)
189
[2-(6-Benzyloxycarbonylamino-purin-9-yl)-acetyl]-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl]-amino}-acetic acid
(4.6d)
Methyl N-(2-Boc-aminoethyl) glycinate (4.7)
190
191
(2-tert-Butoxycarbony -(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin- cetyl]-amino}-acetic acid
methyl ester (4.8a)
lamino-ethyl)-[21-yl)-a
192
mino-2-[2-(4-Benzyloxycarbonyla oxo-2H-pyrimidin-1-yl)-acetyl]-(2-tert-butoxycarbonylamino-ethyl)-amino]-acetic acid
methyl ester (4.8b)
193
2-(5-Benzyloxycarbonylamino-7-oxo-6,7-dihydro-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-acetyl]-(2-tert-
(4.8c) butoxycarbonylamino-ethyl)-amino]-acetic acid methyl ester
194
2-(6-Benzyloxycarbonylamino-purin-9-yl)-acetyl]-(2-tert-butoxycarbonyla r mino-ethyl)-amino]-acetic acid methyl este
(4.8d)
195
2-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-acetyl]-(2-tert-butoxycarbonylamino-ethyl)-amino]-acetic acid
(4.9b)
196
[ -ac d
[2-(2-Benzyloxycarbonylamino-6-oxo-1,6-dihydro-purin-9-yl)etyl]-(2-tert-butoxycarbonylamino-ethyl)-amino]-acetic aci
(4.9c)
197
198
199
4.15b
4.15c
200
N-tert-butoxycarbonyl-3- romopropylamine (4.17) b
3-(2-(2-(2-Methoxy-ethoxy)-ethoxy)-ethoxy)-propyl)-carbamic acid tert-butyl ester (4.19)
201
3-{2-[2-(2-Methoxy ethoxy) ethoxy-]-ethoxy}-propylamine bromo acetamide (4.21)
202
203
204
About the Author
Sridhar Reddy Kaulagari was born in Korpole, Andhra Pradesh and
grew up in a neighboring small town. After graduating from Junior
college he attended Tara Degree College, Osmania University,
Sangareddy, where he majored in natural sciences and earned his
B.Sc and then went to the Osmania University main campus at
Hyderabad where he earned his B.Ed in biological sciences. In the
summer of 2000 he began his graduate studies at the University of
Hyderabad, Hyderabad where he joined the lab of Professor. Ashwini
Nangia to do project as part of the requirements to earn degree in the
School of Chemistry and received his Master’s degree in Chemistry in
2002. He worked as lecturer at Narayana Junior College and MJ
College in Hyderabad for one year. He then moved to Bangalore and
worked as a junior research associate at Cadila Pharmaceuticals Ltd.
In the Fall of 2004, he began his research at the University of South
Florida, Tampa, where he joined the lab of Professor Mark L.
McLaughlin in the Department of Chemistry and the Moffitt Cancer
Center and Research Institute. He will receive his Ph.D in organic
chemistry with a focus on small molecules synthesis in December
2010.